- Email: [email protected]
Rapid detection and identification methods for Listeria monocytogenes in the food chain – A review
Accepted Manuscript Rapid detection and identification methods for Listeria monocytogenes in the food chain – a review Anna-Liisa Välimaa, Anu Tilsala-Timisjärvi, Elina Virtanen PII:
S0956-7135(15)00130-9
DOI:
10.1016/j.foodcont.2015.02.037
Reference:
JFCO 4328
To appear in:
Food Control
Received Date: 16 September 2014 Revised Date:
26 January 2015
Accepted Date: 3 February 2015
Please cite this article as: Välimaa A.-L., Tilsala-Timisjärvi A. & Virtanen E., Rapid detection and identification methods for Listeria monocytogenes in the food chain – a review, Food Control (2015), doi: 10.1016/j.foodcont.2015.02.037. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Rapid detection and identification methods for Listeria monocytogenes in the food
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chain – a review
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Anna-Liisa Välimaaa,1, Anu Tilsala-Timisjärvib,1 and Elina Virtanenb
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a
New business opportunities, National Resources Institute Finland (LUKE), Oulu, Finland
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b
Green technology, National Resources Institute Finland (LUKE), Oulu, Finland
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These authors contributed equally to this work.
8 9 Corresponding author:
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Anna-Liisa Välimaa
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National Resources Institute Finland (LUKE)
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P.O. Box 413
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FI-90014 University of Oulu
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Finland
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email: [email protected]
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phone: +358-40 195 8286
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Abstract
20 Listeria monocytogenes is a facultative pathogenic saprophyte. It can cause a severe disease, listeriosis,
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which is currently considered to be one of the leading food-borne diseases worldwide. L. monocytogenes
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can be found in raw and processed foods. Particularly ready-to-eat (RTE) foods are sources of Listeria
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infections. RTE foods have a long shelf life, because they are stored at low temperatures and in vacuum or
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modified atmosphere packages. Additionally, they are usually consumed without any additional cooking. As
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L. monocytogenes can multiply over a wide range of pH and osmolarity, at low temperatures, and both
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under aerobic and anaerobic conditions, this is a particular concern and necessitates control along the food
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chain. A wide variety of culture and alternative methods have been developed in order to detect or
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quantify this pathogen in food. Here are presented the most rapid and sensitive methods (< 48 h) found in
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the literature that have been used with artificially and/or naturally contaminated food samples. In addition
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to being much more rapid, many of them were as sensitive as the standard methods. However, many
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methods still need to be more thoroughly validated.
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Keywords: Listeria monocytogenes, food, detection, rapid, alternative, methods
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1. Introduction
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Listeria monocytogenes is considered a human pathogen. It has been isolated from urban and natural
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environments, animals and humans (Orsi, den Bakker, & Wiedmann, 2011), and food and food processing
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plants including abattoirs and smokehouses (Moretro & Langsrud, 2004).
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L. monocytogenes can grow over a temperature range from -0.4 °C to 45 °C (Junttila, Niemelä, & Hirn, 1988;
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Walker, Archer, & Banks, 1990), over a pH range from 4.0 to 9.6 (optimum 6–8) (Farber & Peterkin, 1991),
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at water activity (aw) levels of even 0.90 (Farber, Coates & Daley, 1992), and both under aerobic and
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anaerobic conditions. Moreover, L. monocytogenes is able to adhere to a variety of food contact surfaces,
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processing facilities for several months or even years (Miettinen, Björkroth, & Korkeala, 1999; Norton et al.,
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2001; Orsi et al., 2008) as biofilms (Pereira da Silva & Pereira da Martinis, 2013). Protected in biofilms it can
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tolerate high concentrations of many environmental agents, such as sanitizers, disinfectants, and
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antimicrobials, which may result in contamination of food contact surfaces (Carpentier & Cerf, 2011).
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L. monocytogenes may occur in raw foods and in processed foods that are contaminated during and/or
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after processing (EFSA, 2013a). Particularly ready-to-eat (RTE) foods, such as fishery products, heat-treated
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meat products, and cheese, are often sources of Listeria infections. RTE foods can have a long shelf life.
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They are stored at low temperatures and in vacuum or modified atmosphere packages, and are usually
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consumed without any additional cooking. L. monocytogenes has also been found in plant food, e.g. salted
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mushrooms, broccoli, coleslaw, and cantaloupe (EFSA, 2013b). Its ability to grow at low temperatures and
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both under aerobic and anaerobic conditions and in modified atmosphere packaging (Swaminathan &
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Gerner-Smidt, 2007) makes it a great concern for the food industry and necessitates control along the food
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chain (Lambertz, Ivarsson, Lopez-Valladares, Sidstedt, & Lindqvist, 2013) in order to prevent listeriosis, a
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severe threat to public health.
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The first well-documented outbreak of foodborne listeriosis was reported in Canada in 1983, with coleslaw
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as the implicated food (Schlech et al., 1983). Since then, several other outbreaks have been reported. One
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of the deadliest occurred in 2011 in the United States of America (USA); it was associated with cantaloupe
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and caused 146 invasive illnesses, one miscarriage and 30 deaths (Laksanalamai et al., 2012). The risk
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groups for listeriosis are pregnant women, infants, the elderly, and people with weak immune systems;
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among them, it may cause abortion or stillbirth, sepsis, pneumonia or meningitis and serious infections of
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the nervous system, depending on the risk group (Todd & Notermans, 2011).
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L. monocytogenes can access the human food chain directly or through farm animals (zoonotic disease)
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(Gahan & Hill, 2014). The infective dose is suspected to be high; contamination levels in food responsible
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for listeriosis cases are typically >104 CFU/g (Ooi & Lorber, 2005; Vázquez-Boland et al., 2001). Consuming
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foods that contain low levels (<102 CFU/g) of L. monocytogenes is unlikely to cause clinical disease (Chen,
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immunocompromised population (McLauchlin, Mitchell, Smerdon, & Jewell, 2004; Vázquez-Boland et al.,
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2001).
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The case-fatality rate of listeriosis can be high, 20–30% (Todd & Notermans, 2011). In the EU, 1476
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confirmed cases were reported in 2011. Of all the zoonotic diseases under EU surveillance, listeriosis
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caused the most severe human disease with a fatality rate of 12.7% (EFSA, 2013c).
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In the USA, 1600 listeriosis cases are estimated yearly, with 250 deaths (Scallan et al., 2011). The total
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economic cost of listeriosis, comprising health care costs, lost productivity, and diminished quality of life, is
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estimated to amount to almost USD 2.04x1012 and the cost per case to nearly USD 1,282,000 annually
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(Byrd-Bredbenner, Berning, Martin-Biggers, & Quick, 2013). Sales reductions of RTE foods due to L.
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monocytogenes recalls were estimated to be 22–27% after the event (Thomsen, Shiptsova, & Hamn, 2006).
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Legislation addressing L. monocytogenes contamination in food varies. In the EU, for a healthy human
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population, foods not exceeding the limit of 100 CFU/g are considered safe (EC, 2005). If the foods are able
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to support L. monocytogenes growth and the food processor cannot demonstrate that this limit is not
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exceeded during the shelf life, L. monocytogenes must be absent. In any case, in RTE products intended for
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infants and for special medical purposes, L. monocytogenes must be absent in 25 g. In the USA, the Food
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and Drug Administration (FDA) has a zero tolerance policy for L. monocytogenes in RTE products (FDA,
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2003); that said, in a nonbinding compliance policy guide, the L. monocytogenes level was set at <100
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CFU/g for RTE foods that do not support its growth (FDA, 2008).
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Here, rapid methods used for the detection of L. monocytogenes in food are presented, especially in the
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context of the production environment and finished products. The recently published literature describing
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L. monocytogenes detection, quantification or identification in naturally or artificially contaminated food
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samples is reviewed. Market report information and other data on the usage of microbiological detection
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methods in the food industry are included, focusing on Europe and the USA. Patent information is also
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covered. The aim is to give an overview of selected current rapid (<48 h) L. monocytogenes detection or
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identification methods and bring them to the awareness of all interest groups in the food production field
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who are involved in ensuring food safety.
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2. Detection and quantification methods for L. monocytogenes and Listeria spp. in food
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Various microbiological methods have been used for decades in the food industry for the detection of
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bacteria. Legislation in the EU and USA still relies on methods based on culture assays. These methods are
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designed to enable the detection of a single target cell in a sample, and this is also the desired
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characteristic of any rapid method intended to replace a culture-based one (Dwivedi & Jaykus, 2011). As
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“rapid” may mean a shorter time, but can also refer to better flow-through or handling of multiple samples
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for greater convenience and automation of work, Jasson, Jacxsens, Luning, Rajkovic, and Uyttendaele
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(2010) suggested using the term “alternative method”. Wiedmann, Wang, Post, and Nightingale (2014)
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have thoroughly outlined criteria for evaluation of rapid detection methods in the food industry, which may
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help in the selection of a suitable one. Microbiological methods can be classified as quantitative or
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qualitative. Quantitative methods (enumeration) aim at determining the number of bacteria present,
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directly or indirectly, in a given sample. Qualitative methods will demonstrate whether the target
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bacterium is present or not in the sample.
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If a food sample also contains other Listeria species, particularly L. innocua, L. monocytogenes may be
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overgrown by them, which may lead to false negatives (Gnanou Besse et al., 2010). However, when Listeria
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spp. is targeted, a positive result may also indicate presence of L. monocytogenes.
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2.1. Classical cultural methods
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Classical detection methods employing enrichment-plating techniques are used for the testing of presence
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or absence of pathogens, usually in 25 g of food, and the overall detection limit (DL) is ca. 1–5 CFU/test
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portion (Jasson et al., 2010).
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salts, preservatives, and other chemicals or natural antimicrobial compounds (Wu, 2008). Additionally,
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viable bacteria may respond to various environmental stresses by entering a dormancy state where the
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cells remain viable, but non-culturable (VBNC) on standard laboratory media (Oliver, 2010). If resuscitated
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from this VBNC state, the cells can again be cultured and cause infection. Therefore, their recovery during
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culturing procedures is critical.
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Due to these sub-lethally injured and VBNC states, the detection method is often divided into a two-step
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enrichment process. The pre-enrichment step in a non- or half-selective medium resuscitates the injured
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target organisms and increases them. It also dilutes inhibiting compounds and rehydrates bacterial cells
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derived from dried or processed food matrices (Dwiwedi & Jaykus, 2011). The second enrichment step in a
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selective medium (containing e.g. different salts and antibiotics) suppresses the background flora and
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increases the target pathogen, resulting in a million-fold multiplication of the target, enabling its isolation
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or detection (Jasson et al., 2010). Presumptive positive colonies are isolated on a selective differential agar
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medium. If typical colonies are absent, the analysis is completed. The presumptive colonies are confirmed
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by additional morphological, biochemical, physiological, and/or serological testing. It can take up to 4 days
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to detect the pathogen presumptively (appearance of typical colonies on the medium) or to get a negative
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result (no typical colonies), and confirmation of the positive result can take around one week (Dwiwedi &
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Jaykus, 2011; Jasson et al., 2010).
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Various official culture-based L. monocytogenes detection methods (e.g. International Organization for
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Standardization (ISO) and FDA), and their differences have been outlined by Zunabovic, Domig, and Kneifel
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(2011). They are recommended for or have been established with different target matrices, and they use
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different pre-enrichment media and incubation times, whereas the incubation temperature is the same (30
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°C). The selective enrichment media, incubation temperature (30/35/37 °C), and time vary according to the
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standard.
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Convenience methods are conventional microbial analysis methods that have been modified or automated
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to make them faster and less laborious, e.g. using fluorogenic or chromogenic substrate in selective media
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and avoiding the need for sub-culturing and further biochemical testing (Mandal, Biswas, Choi, & Pal,
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2011). Commercial morphological, biochemical, and physiological tests are also available for identifying
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pathogens using conventional methods.
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The (alternative) diagnostic methods often combine various technologies (culture, immunoassays, nucleic
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acids). Table 1 presents the detection limits and enrichment times of the standard and some alternative
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methods for Listeria monocytogenes determination in food. The limit for ISO 11290 is based on various
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validation certificates or reports available at the AFNOR NF Validation website (http://nf-
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validation.afnor.org/en/alternative methods/). The rapid methods (≤ 48 h) found in the literature are
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included here if their reported detection limit is ≤104 cells and they have been tested in food. The DL can be
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given as the limit in the food sample before enrichment or as the limit for the molecular test (Wiedmann et
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al., 2014). Thus, because the DLs of the methods are presented in various meanings or units in the
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literature, it is not possible to make direct comparisons.
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2.2.1. Immunoassays
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Immunological assays are based on the specific binding of an antibody and an antigen. The capturing part
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of the antigen recognizes the epitopes on the antibody and binds to them. A monoclonal antibody may
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identify a particular epitope whereas a polyclonal antibody may recognize several epitopes of a certain
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antigen or many antigens. The DL is influenced by the antibody and the immunological assay type (about
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103–107 CFU/ml in the literature). In order to achieve the set detection limit criterion, enrichment is usually
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needed. The average sensitivity of immunological methods has been improved in the examples given here.
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The most widely used immunological methods in food diagnostics are lateral flow immunoassay, enzyme-
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linked immunosorbent assay (ELISA), enzyme-linked fluorescent assay (ELFA), and immunomagnetic
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separation (IMS). IMS is included in many detection methods for separating and concentrating the target
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sample matrix.
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In lateral flow immunoassay or immunochromatography the reaction between an antibody and its target is
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detected visually. It relies on the migration of the target in a sample to the antibody that is immobilized on
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a membrane surface. A lateral flow chromatographic enzyme immunoassay together with a magnetic
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concentration step was demonstrated to detect 102 CFU/ml L. monocytogenes in spiked milk within two
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hours (Cho & Irudayaraj, 2013) with monoclonal L. monocytogenes-specific antibodies LZF7 and LZH1.
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Blažková, Koets, Rauch, and van Amerongen (2009) combined polymerase chain reaction (PCR) and lateral
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flow immunoassay for detecting the amplified products for Listeria spp. and L. monocytogenes. An aliquot
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of the PCR products was added to an assay device, and the presence of a specific amplicon was visualized
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with a dark line. It detected <10 cells/25 ml milk within 28 hours including 24 h enrichment, and was tested
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with 24 authentic food samples and found to be in concordance with the ISO standard method. Kovačević
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et al. (2009) evaluated commercial assays for identifying Listeria spp. at meat processing facilities with
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environmental samples from various production stages. A lateral flow assay was found to be as specific as
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culturing and a PCR assay and not significantly less sensitive with >300 tested samples.
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In ELISA an immunological and an enzyme assay are combined, usually in a sandwich ELISA form. Shim et al.
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(2008) developed an immunochromatography (ICG) strip test and an ELISA test with IMS (3B12-17 MAb) for
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qualitative detection of Listeria spp. and L. monocytogenes. They were used with 116 naturally
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contaminated meat samples, and ICG-IMS could detect these pathogens in 15 hours, with a limit of 102
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CFU/10 g. An ELISA test targeting L. monocytogenes in meat, seafood, and dairy products was validated
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against the ISO method by Portanti et al. (2011) using 190 naturally and 30 artificially contaminated
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samples. It used a single enrichment step and produced results in concordance with the standard. The DL
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was 6.6 x 103 CFU/ml and the relative DL 5–10 CFU/g in spiked samples.
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In order to enhance the sensitivity of ELISA, a fluorescent label can be added to the antibodies (ELFA). A
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time-resolved fluorescent immunoassay for Listeria spp. (Jaakohuhta, Harma, Tuomola, & Lovgren, 2007)
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was compared to a commercial ELFA test by measuring 22 naturally contaminated cheese, fish, milk,
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and was more sensitive (20 CFU/ml). A commercial ELFA, a commercial RT-PCR designed for food, and
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culturing were evaluated for detecting L. monocytogenes in 50 vacuum-packed meat products (Netschajew,
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Fredriksson-Ahomaa, Sperner, & Stolle, 2009). Immunoassay and culture found only some positives, while
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the RT-PCR detected 32 as positive after an overnight enrichment. The difference was explained by a lower
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contamination level or high number of unculturable cells.
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Different types of fluorescent systems have been developed for increasing the sensitivity of immunoassays.
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L. monocytogenes was targeted simultaneously with E. coli and S. typhimurium using magnetic nanobeads
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with polyclonal antibodies for separation and quantum dot (QD) fluorescence for detection in 2 hours
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(Wang, Li, Wang, & Slavik, 2011). A DL of 20–50 CFU/ml in inoculated ground beef, chicken, broccoli, and
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lettuce samples was reported. Cho, Mauer, and Irudayaraj (2014) developed a slightly more sensitive
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method applying immunomagnetic beads with L. monocytogenes-specific monoclonal antibodies for
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capture and antibody-BSA labelled with fluorophores for detection. The feasibility of the L. monocytogenes
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system was demonstrated with spiked milk in 4 hours (including a 2 h incubation at RT), detecting <5
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CFU/ml of L. monocytogenes. A multiplex detection with E. coli and S. typhimurium (mixed culture samples)
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was also demonstrated. Other Listeria species were not included for testing in either of the above multiplex
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methods.
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2.2.2. Biosensors
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A biosensor is an analytical device converting a biological response into an electrical signal. It consists of a
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bioreceptor (microorganism, cell, enzyme, antibody, nucleic acid) and a transducer (Velusamy, Arshak,
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Korostynska, Oliwa, & Adley, 2010). The transducer may be optical (Raman/Fourier transform IR-
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spectroscopy, surface-plasmon resonance, optic fibres), electrochemical (amperometric, impedimetric,
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potentiometric, conductometric), mass-based (piezoelectric, magnetic) or thermal. Biosensors have been
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recently reviewed in the context of food pathogens (e.g. Arora, Sindhu, Dilbaghi, & Chaudhury, 2011; Arora,
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Sindhu, Kaur, Dilbaghi, & Chaudhury 2013; Sharma & Mutharasan, 2013a). Truly rapid biosensor methods
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Rogers, and Suni (2013), who used impedance spectroscopy and monoclonal IgG1 antibodies, and achieved
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a calculated DL of 4 CFU/ml for L. monocytogenes in an artificially inoculated tomato extract without
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enrichment.
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Davis et al. (2013) used screen-printed carbon electrode (SPCE) strips as disposable amperometric sensors
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for ELISA with commercial anti-L. monocytogenes antibodies and demonstrated detection of 102 CFU/g L.
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monocytogenes from spiked blueberries in about one hour. Instead of an electrochemical transducer,
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Sharma and Mutharasan (2013b) demonstrated L. monocytogenes detection in milk using a mass-based
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piezoelectric cantilever biosensor and a commercial polyclonal anti-L. monocytogenes antibody, with a limit
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of 103 cells/ml. Adding a third antibody binding step into the method enabled the detection of 102 cells/ml
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in one hour.
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A fibre-optic immunosensor system based on sandwich immunoassay with MAb C11E9 and fluorescence
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detection was developed by Geng, Morgan, and Bhunia (2004). They demonstrated that this system
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detected L. monocytogenes in artificially or naturally contaminated hot dogs and bolognas, in 24 hours
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after 20 h enrichment (10–103 cells/g inoculations). An oligonucleotide aptamer specific for internalin A
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was used as a reporter molecule in a fibre-optic biosensor (Ohk, Koo, Sen, Yamamoto, & Bhunia, 2010). The
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DL of the assay was 103 CFU/ml, and it could detect 4 CFU/g spiked L. monocytogenes in deli meats (beef,
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chicken, and turkey) after 18 h enrichment. The type of enrichment broth used influenced the success of
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the detection. A DL of 3 x 102 CFU/ml was obtained for L. monocytogenes by a fibre-optic sensor together
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with immunomagnetic cell separation using anti-InlA MAb-2D12 antibodies (also specific for L. ivanovii)
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(Mendonҫa et al., 2012). In soft cheese and hotdogs 10–40 CFU/g were detected after 18 h enrichment.
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The total analysis time of these example methods is < 24 h or only a few hours, but in general their DLs are
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higher than those of amplification methods, so their sensitivity needs improvement. Some methods also
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need further validation with reference species and different types of food samples.
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2.2.3. Bacteriophage-based detection methods
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Koeris, 2013; Singh, Poshtiban, & Evoy, 2013; Smartt et al., 2012). The mentioned advantages of phage-
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based detection include target specificity, sensitivity, distinction of live and dead cells, and ease of detector
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amplification. Commercial immunoassays using bacteriophage tail proteins for detecting Listeria and L.
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monocytogenes are available. A system utilizing phages to produce illuminative reporter proteins in Listeria
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cells is also on the market. In the literature, phage-based proteins have been applied for the separation and
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detection of Listeria through the use of paramagnetic beads and culturing (Kretzer et al., 2007). The
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bacteria were detected from spiked and 275 naturally contaminated food samples (lettuce, cheese, salmon,
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meat, milk). A limit of 102 CFU/g was obtained in all food materials after 6 h pre-enrichment and a limit of
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0.1 CFU/g in all categories except soft cheese after 24 h enrichment. The sensitivity was better than with
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the standard culture method. Use of these phage protein-coated beads for separating L. monocytogenes
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directly from artificially contaminated raw milk samples and detecting them by plating or RT-PCR was
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evaluated by Walcher et al. (2010). The achieved DL by IMS, DNA-isolation, and RT-PCR (on prfA;
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transcriptional activator of the virulence factor) was 102–103 CFU/ml.
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2.2.4. Fluorescent in situ hybridization (FISH)
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Ribosomal RNA targeting probes are commonly used with FISH, where the hybridization is detected by
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fluorescence microscopy. FISH in the context of food diagnostics has been recently reviewed by Rohde,
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Hammerl, Appel, Dieckmann, and Al Dahouk (2015). Detection of viable Listeria spp. (23S rRNA probe) or L.
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monocytogenes (16S rRNA probe) cells from spiked smoked salmon, camembert and mozzarella cheese,
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ham, and cabbage samples (102–3x103 CFU/ml) has been carried out using FISH with filter cultivation
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(Fuchizawa, Shimizu, Kawai, & Yamazaki, 2008; Fuchizawa, Shimizu, Ootsubo, Kawai, & Yamazaki, 2009).
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The hybridization was observed by fluorescent microscopy. The method was equal to a plating method in
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enumerating the cells but took less time (enrichment and FISH 12–16 h in total). Viable L. monocytogenes
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were detected from 191 fresh and frozen vegetable samples by culture (ISO Standard), PCR targeting 16S
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rDNA and hlyA for Listeria genus and L. monocytogenes, and Direct Viable Count (DVC)-FISH targeting L.
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monocytogenes 16S rRNA (Moreno et al., 2012). In DVC bacteria are grown in a medium that prevents cell
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division and promotes elongation of cells with metabolic activity, which are counted as viable. With culture,
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PCR, or DVC-FISH 4%, 10%, or 33% of the samples were positive, respectively. DVC-FISH enabled the
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detection of the viable cells (7.4x102–9.4x104 CFU/g) after 7 h culturing.
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In the literature, various amplification methods are the most widely used alternative methods for L.
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monocytogenes detection in food samples. Quantitative PCR (qPCR) or real-time PCR (RT-PCR) application
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in food microbiology and diagnostics has been reviewed earlier (Cocolin, Rajkovic, Rantsiou, & Uyttendaele,
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2011; Postollec, Falentin, Pavan, Combrisson, & Sohier, 2011), likewise the use of multiplex-PCR for
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targeting food microbes (Settanni & Corsetti, 2007). Other amplification methods have emerged, e.g.
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isothermal amplification (including loop-mediated isothermal amplification (LAMP) and hyperbranching
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rolling circle amplification (HRCA)), and nucleic acid sequence-based amplification (NASBA) for amplifying
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RNA templates. An overview of current technologies for isothermal amplification has been presented by
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Craw and Balachandran (2012.)
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2.2.5.1. Conventional PCR
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Conventional qualitative or semi-quantitative PCR relies on the endpoint analysis of the amplified products
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(electrophoresis, fluorescence). A commercial, non-validated Listeria monocytogenes detection kit was
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evaluated on spiked, enriched (24 h) raw pork sausage and mozzarella cheeses (Amagliani, Giammarini,
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Omiccioli, Brandi, & Magnani, 2007), and it detected 1 CFU/g L. monocytogenes. While optimizing an
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internal control (IAC) for a L. monocytogenes PCR-assay, Rip and Gouws (2009) detected 8 CFU/ml from
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spiked and enriched (22 h) camembert cheese and ostrich meat samples targeting the hly gene. Delibato et
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al. (2009) have described a combined PCR and microfluidic chip-based automated electrophoresis system
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for detecting L. monocytogenes in 50 naturally contaminated food samples (RTE meat, cheese, smoked
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of positive samples.
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Multiplex PCR systems enable the detection of different species at the same time in one run or different
303
targets in one reaction. A system for identifying viable Listeria genus and different Listeria species (L.
304
monocytogenes, L. innocua, and L. grayi) was developed using reverse transcription-based PCR targeting
305
iap mRNA (Rattanachaikunsopon & Phumkhachorn, 2012). The DL was 50 CFU/ml in pure cultures. The
306
feasibility of the system was evaluated with spiked meat samples (102 CFU/ml) with one-hour enrichment.
307
Zeng, Zhang, Sun, and Fang (2006) used multiplex-PCR for detecting Listeria spp. and L. monocytogenes iap
308
and hly genes in samples from spiked milk and isolates from a milk processing environment (raw milk,
309
sewage water, vessel surfaces) to contain L. monocytogenes. PCR and API identified the same isolates. The
310
DL of the PCR was 1.4 x 102 CFU/ml directly in spiked milk but an enrichment of 3–6 h yielded a limit of 1.45
311
CFU/ml. Multiplex-PCR for L. monocytogenes (prfA gene), Salmonella spp., and E. coli was demonstrated in
312
liquid egg samples (Germini, Masola, Carnevali, & Marchelli, 2009). The assay detected 10 cells in 25 g after
313
15 h enrichment. L. monocytogenes was also included in simultaneous detection of multiple food
314
pathogens from spiked milk and chicken meat samples by a chromogenic macroarray system, where biotin-
315
labelled PCR products were probed for further sensitivity (Chiang et al., 2012). A HtpG-like gene was used
316
as a target for L. monocytogenes. In 18 hours the system could detect 1 CFU/ml or g after an 8-h
317
enrichment step. This level was also achieved directly by PCR, but macroarray increased the sensitivity
318
when enrichment was not used. Naturally, the L. monocytogenes primers of the systems above could be
319
used separately for detecting only this species. The value of multiplexing might be limited by the different
320
enrichment (media) needs of target pathogens and by reduced sensitivity compared to using single primer
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pairs (Wiedmann et al., 2014).
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2.2.5.2. Real-time PCR
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In real-time PCR the formation of amplification products can be monitored based on fluorescence as an
325
endpoint analysis or in real time. Another advantage over conventional PCR is the ability to quantify the
14
ACCEPTED MANUSCRIPT start material. RT-PCR is also faster, and it is widely used in methods targeting food pathogens. An example
327
of endpoint analysis is identification of L. monocytogenes and five other Listeria species by RT-PCR (ssrA
328
gene) and high-resolution melting curve analysis (Jin et al., 2012). The sensitivity with L. monocytogenes
329
inoculated food samples was 102 CFU/ml, and the method was used for 30 artificially contaminated
330
samples (juice, milk, cheese, and meat) with a 93.3% correction rate.
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Real-time analysis is more common. A combined 24 h enrichment and RT-PCR method for L.
332
monocytogenes targeting the prfA gene gave DLs of 7.5 CFU/25 ml and 1–9 CFU/15 g in artificially
333
contaminated raw milk, and salmon, pâté, and cheese, respectively (Rossmanith, Krassnig, Wagner, & Hein,
334
2006). With 76 naturally contaminated food samples (fish; meat; meat and dairy products) 96% accuracy,
335
100% specificity and 76.9% sensitivity were achieved compared to the ISO standard. The method was also
336
applied for confirming the plating results during a multinational listeriosis outbreak (Schoder, Rossmanith,
337
Glaser, & Wagner, 2012). RT-PCR (actA gene) preceded by 24+6 h enrichment was evaluated against the
338
standard ISO method with 144 naturally and 61 artificially contaminated meat, fish, dairy, and vegetable
339
samples (Oravcová, Kuchta, & Kaclikova, 2007). Contamination of 1 CFU/25 g was detected, also from three
340
artificially contaminated samples that were negative with the ISO method. Enrichment (24+8 h) and RT-PCR
341
(here for ssrA) were also used for detecting L. monocytogenes in 175 samples taken from retail outlets and
342
food processing plants (O’Grady et al., 2009). The method had 99.44% specificity, 96.15% sensitivity and
343
99.03% accuracy compared to the ISO standard, and the DL was 1–5 CFU/25 g.
344
Rantsiou, Alessandria, Urso, Dolci and Cocolin (2008) developed and used L. monocytogenes qPCR (16S-23S
345
intergenic spacer) for 66 authentic food samples. Some positive results after 24 h enrichment were not
346
confirmed by cultural tests, which were interpreted as false negative. This was explained by the use of a
347
non-official isolation method and by competition from other species during the culture. Quantification limit
348
was 103–104 CFU/ml directly, but 10 CFU/ml could be detected after overnight enrichment. This RT-PCR was
349
also compared with the modified ISO method for the occurrence of L. monocytogenes in a dairy processing
350
plant (Alessandria, Rantsiou, Dolci, & Cocolin, 2010). Investigation of 200 samples (fresh cheeses, brine, and
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environmental samples where cells may be under stress conditions and possibly in VBNC state.
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Use of a L. monocytogenes (hly gene) RT-PCR detection method developed earlier (Rodríquez-Lázaro et al.,
354
2004; Rodríquez-Lázaro, Pla, Scortti, Monzó, & Vázquez-Boland, 2005) has recently been evaluated or
355
validated in several food matrices. A validation trial of RT-PCR-based L. monocytogenes detection in
356
artificially contaminated soft cheese was reported by Gianfranceschi et al. (2014). Results from 11
357
European laboratories showed that RT-PCR with ISO compatible enrichment and DNA extraction performed
358
significantly better than the reference method (less false negatives and positives). It detected 10 CFU/25 g
359
in 27 h, and the relative accuracy was 82.75%, relative specificity 96.70%, and relative sensitivity 97.62%.
360
Also, the presence of L. innocua had a greater effect on the results obtained with the reference method
361
than by RT-PCR. In another study Rodríquez-Lázaro, Gonzalez-García, Gattuso, Gianfranceschi, and
362
Hernandez (2014) used this L. monocytogenes RT-PCR for artificially and naturally contaminated pork meat,
363
poultry meat, RTE lettuce salad, and sheep milk cheese samples. The best result was achieved when a 25 g
364
sample was diluted 1:10, enriched for 24 hours and DNA extracted using a commercial silica column. It
365
detected 2-4 CFU/25 g in 27 hours. Analytical performance with 200 natural samples was similar to that of
366
the reference method.
367
Some examples report that combining IMS and RT-PCR improves detection sensitivity. Use of nanoparticles
368
coated with anti-L. monocytogenes antibodies and RT-PCR (hlyA gene) was evaluated for detecting L.
369
monocytogenes in artificially contaminated milk (Yang, Qu, Wimbrow, Jiang, & Sun, 2007). Nanoparticles
370
performed better compared to Dynabeads, the sensitivity of the method being 226 CFU/0.5 ml. Duodu,
371
Mehmeti, Holst-Jensen, & Loncarevic (2009) evaluated filtration and IMS (Dynal anti-Listeria) in
372
combination with RT-PCR (hlyA) to detect L. monocytogenes in artificially contaminated hot-smoked
373
salmon. IMS reduced PCR inhibition significantly, and the DL was 20–40 CFU/g in a total of 3.5 h without
374
enrichment.
375
Several examples report differentiation of dead and live L. monocytogenes cells by RT-PCR. It was used for
376
differentiating viable (also VBNC) and dead L. monocytogenes cells in gouda-like cheese (Rudi, Naterstad,
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ACCEPTED MANUSCRIPT Dromtorp, & Holo, 2005). Ethidium monoazide bromide treatment enabled quantification of live vs. dead
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cells in spiked samples; 16–20 h enrichment and RT-PCR allowed detection of 10 CFU/g. Effect of
379
bacteriocin treatment on detecting L. monocytogenes by qPCR targeting hly was investigated using spiked
380
mixed salad samples (Cobo Molinos, Abriouel, Ben Omar, Martinez-Canamero, & Galvez, 2010). Above the
381
threshold level of 102 CFU/g the assay was better than the plating method for evaluating live cells but
382
below this level enrichment should be included. A filtering pre-treatment combined with quantitative RT-
383
PCR of the prfA gene enabled detecting 10 viable cells of L. monocytogenes in 10 g of spiked yogurt (D’Urso
384
et al., 2009) without enrichment. The method was in agreement with the ISO standard. Also Ye et al. (2012)
385
detected viable L. monocytogenes (1 CFU/ml) without enrichment in artificially contaminated frozen pork
386
by reverse transcriptase RT-PCR (hly). The method was recommended for showing the presence of live cells
387
but only for rough quantification of contamination.
388
RT-PCR has also been used in multiplex systems. An analysis consisting of enrichment (16–20 h), DNA
389
extraction and RT-PCR with dual-labelled probes or high resolution melting analysis was developed by
390
Omiccioli, Amagliani, Brandi, and Magnani (2009). It detected 1 CFU of each species – L. monocytogenes
391
(hlyA gene), E. coli, and Salmonella spp. – in 125 ml of spiked milk (divided into 25 ml aliquots). A multiplex
392
RT-PCR targeting L. monocytogenes (hly gene) and Salmonella spp. was described by Ruiz-Rueda, Soler,
393
Calvo, and Garcia-Gil (2011), and evaluated for L. monocytogenes with 54 naturally (dairy products;
394
vegetable, fish and meat matrices; RTE food) and artificially contaminated samples in <30 h. Sensitivity was
395
94.1% and efficiency 94.4% for L. monocytogenes compared to the ISO standard. The DL was 5 CFU/25 g
396
(for eggs and smoked salmon 102 CFU/25 g). Another assay for L. monocytogenes and Salmonella spp. was
397
developed by Garrido et al. (2013) and used for 63 spiked and 95 natural samples (fish, vegetables,
398
seafood, RTE, by-products, environmental). The detection limit was 5 CFU/25 g after 24±2 h and 8 h
399
enrichments, and the quality parameters >90%. A simultaneous enrichment (18 h) and detection of L.
400
monocytogenes (2 CFU/g) with Campylobacter, Salmonella, and E. coli in 24 h was developed and used for
401
212 routine samples (Köppel, Kuslyte, Tolido, Schmid, & Marti, 2013) in concordance with the ISO method.
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Terminology concerning the performance limits of the methods varies. Postollec et al. (2011) point out that
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the DL means the minimum number of microbes that can be detected, but the quantification limit is the
404
lowest amount that can be accurately quantified and is usually higher than DL. Also, the limits obtained in a
405
food matrix should be used instead of limits in pure cultures.
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Most isothermal amplification-based methods for Listeria employ the LAMP technique, which uses several
409
primer pairs in a reaction performed at constant temperature, avoiding the need for special instruments.
410
Compared to PCR and RT-PCR other advantages include high specificity, speed and tolerance of sample
411
impurities (Niessen, Luo, Denschlag, & Vogel, 2013). Also, RNA can be used directly as a target in the
412
reaction for increased sensitivity.
413
A LAMP assay for L. monocytogenes was developed by Wang, Huo, Ren, and Li (2010) using primers based
414
on iap (P60 extracellular protein, invasion associated protein IAP) and evaluated with 125 naturally
415
contaminated milk samples. The method was 100% in concordance with the ISO standard when a 6-h
416
enrichment was included. The DL of the assay was 186 CFU/ml or 8-10 cells/reaction. Wan et al. (2012)
417
used propidium monoazide with LAMP based on the hlyA gene for detecting viable L. monocytogenes in
418
spiked chicken, pork, ground beef, and milk powder. The DL of the system was 102 CFU/ml in pure culture
419
and 3.1 x 103 CFU/g in all tested food types. A LAMP assay based on L. monocytogenes prfA together with
420
12/24 h enrichment was used for artificially contaminated milk (Cho, Dong, Seo, & Cho, 2014) and it
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detected 2.22 x 101–2.22 CFU/ml L. monocytogenes. In pure culture the DL was 2.22 x 102 CFU/ml. A
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commercial LAMP kit for Listeria based on sigB sequences (3M Molecular Detection Assay Listeria) was
423
validated against two cultural methods (3M Modified Listeria Recovery Broth and FDA BAM standard
424
method) with 391 environmental sponge samples from retail, and meat, dairy, and seafood processing
425
plants (Fortes, David, Koeritzer, & Wiedmann, 2013). The results after 22 h enrichment were not different
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from the culture methods, and they could be obtained in 24 hours. Other, non-validated LAMP kits are also
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available. Another type of isothermal amplification, hyperbranching rolling circle amplification (HRCA),
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based on hly gene and gold nanoparticle-based colorimetric assay was demonstrated for L. monocytogenes
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detection in artificially contaminated milk (Fu, Zhou, & Xing, 2013). The sensitivity of the assay was 100 aM
430
target gene or 75 copies of genome DNA/reaction. In general, isothermal methods are not yet as sensitive
431
as PCR or RT-PCR, but they are being improved. More thorough validation testing is also needed.
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2.3. Characteristics of various detection methods
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Conventional (culture and chromatographic) techniques are based on phenotypic tests. Their advantages
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include sensitivity, reliability in efficiency, applicability to food samples, and low cost, but a major
437
disadvantage is the long time to result, in addition to labour intensiveness and lack of specificity (Jasson et
438
al, 2010; Yeni, Acar, Polat, Soyer, and Alpas, 2014).
439
The specificity of immunological methods relies on antigen-antibody binding and may not always be high
440
enough. They are also less sensitive than nucleic acid-based methods (Yeni et al., 2014). On the other hand,
441
these tests can be automated and are fast, reproducible, and less sensitive to food interference (Jasson et
442
al., 2010).
443
Compared to the other techniques, nucleic acid-based methods are more specific, using DNA or RNA
444
sequences as targets. RNA instead of DNA provides more sensitivity and information about the viability of
445
the bacteria (Jasson et al., 2010). On the other hand, because rRNA is rather stable and degrades slowly
446
after bacterial cell death, assays targeting rRNA may give false positives whereas mRNA-based tests are less
447
likely to do so (Wiedmann et al., 2014). Molecular tests are rapid and reproducible, enable multi-parameter
448
testing, and can be automated (Jasson et al., 2010; Yeni et al., 2014). The drawbacks include higher costs
449
and the negative effect of the food matrix on performance, especially with PCR (Jasson et al., 2010; Yeni et
450
al., 2014). Yet, Rodriguez-Lavaro et al. (2014) calculated that the RT-PCR method (EUR 3) is cost-effective
451
compared to the standard method (EUR 15). De Medici et al. (2014) also came to the same conclusion,
452
considering all analysis costs.
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have an impact on the final result. Likewise, the type of food, other microbes in food samples, and the
455
stress conditions on the target pathogen (e.g. sub-lethally injured cells) all affect the test performance
456
(Jasson et al., 2010).
457
The most rapid alternative methods have been performed with a food matrix within 1-3.5 hours with the
458
DLs ranging from 10–40 CFU/g to 102-103 CFU/ml without any enrichment (Table 1). The sensitivity of the
459
methods is usually increased by enrichment. Using RT-PCR, a level of 1–5 CFU/25 g after 20–30 h culturing
460
has been reported (Table 1). At this detection level the shortest enrichment times were 3–6 h (Zeng et al.,
461
2006). Also methods without any enrichment steps have been reported.
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The Food Safety and Inspection Service (FSIS) (FSIS, 2014) provides a list of foodborne pathogen test kits
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validated by independent organizations (AOAC, AFNOR, MicroVal or NordVal). Currently over 90 detection
467
methods are listed for Listeria spp. and L. monocytogenes. The same kit can be validated separately for
468
different matrices or purposes (detection, enumeration). One third of the tests target L. monocytogenes.
469
An Internet search was performed for commercially available, but non-validated alternative tests; the
470
results are presented in Table 2. The non-comprehensive list of nearly 40 tests shows that, in addition to
471
the officially approved tests, there exists a wide range of methods for the detection of Listeria, mostly
472
based on some amplification method. Half of them are L. monocytogenes specific.
473
Detection kits based on amplification and hybridization techniques are the most numerous (43%) among
474
the validated Listeria/L. monocytogenes alternative methods, followed by immunological (31%) and culture
475
(26%) methods. Nucleic acid technology, especially RT-PCR, is the most commonly found type also in the
476
case of non-validated commercial tests.
477
The fastest total analysis times given are 16–24 h for the ANSR Listeria test (Neogen Corporation), 20.5 ± 2
478
h for the ADIAFOOD Listeria and L. monocytogenes tests (bioMérieux SA) and 20 ± 2 h for the GeneDisc
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amplification. RapidChek Listeria (Romer Labs) and Vidas LMX Listeria monocytogenes (bioMérieux SA) are
481
immunological tests claimed to yield results in 24 hours. A new Sample6 DETECT/L (Sample6) for Listeria
482
environmental samples has recently received an AOAC certification. The test utilizes bacteriophages and is
483
stated to yield results in < 8 hours without enrichment.
484
485
4. Detection methods used by the food industry
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Although rapid or alternative methods are increasing in number and have been commercially available for
488
many years, it has not been evaluated in the literature how widely they are actually being used in the food
489
industry.
490
Some information is, however, given on the topic. According to a review by The Food Standards Agency in
491
the UK (FSA, 2005) on the microbiological methods used by the food industry or laboratories, the most
492
popular food categories tested were milk and dairy products and meat, poultry, and related products.
493
Conventional standard culture methods were the most common category, out of which the aerobic
494
mesophilic plate count method (30 °C) was used by 84% of the laboratories. The most common externally
495
performed test type was pathogen testing, and of the tests for specific bacteria carried out in-house, the
496
most popular were tests for Staphylococcus aureus and other coagulase positive staphylococci (CPS),
497
Salmonella, Listeria spp., Bacillus cereus, and L. monocytogenes.
498
Only a minority of companies had replaced the traditional methods with alternative ones. The reluctance to
499
adopt new techniques was attributed to the cost of capital equipment and consumables, insufficient
500
validation data, the need to demonstrate the suitability of alternative methods in-house, and lack of
501
knowledge in connection with molecular methods. A majority of 71% of laboratories were not planning to
502
adopt new methods. The report was based on surveys carried out in 1998–2001, so it is assumed that the
503
alternative methods have gained wider usage since then.
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505
Supply Chain, an EU project collecting information on the use of and future needs for analysis methods,
506
including rapid methods, in 11 EU member countries and 6 non-EU members (Lebesi, Dimakou, Alldrick, &
507
Oreopoulou, 2010; Lebesi, Bilbao, Diaz, Papadaki, & Oreopoulou, 2011). The samples examined most often
508
were final products (94% of the respondents) and raw materials (90%), followed by environmental samples
509
(69%) and intermediate products (59%) (Lebesi et al., 2010). The largest respondent groups in terms of
510
product categories were meat and fish, beverages, dairy, and fruit/vegetable companies. Nine per cent
511
performed the analyses only in-house, 35% used only external laboratories, and 56% had tests performed
512
both internally and externally. Microbiological contaminants accounted for 90% of the analytes tested.
513
Of the respondent food companies performing analyses in-house, two-thirds used rapid methods in daily
514
operations, most commonly for microbiological analytes. The most widely used rapid tests were for E. coli
515
(~32%), total bacteria (~23%), and Salmonella (~18%). Rapid Listeria tests were used by about 12% of
516
respondents and 16% stated a need for one (Lebesi et al., 2011). The number of non-validated rapid
517
microbiological methods was presented to be around 160, the number of validated methods more than 50,
518
and the number of rapid methods used by the food industry more than 40.
519
Almost all respondents expressed an interest in extending the range of tests performed, and in terms of
520
their future needs, most ranked rapid microbiological tests as a priority. The introduction of rapid methods
521
was seen as an improvement in food safety follow-up (62% of the respondents). In all, new rapid methods
522
were already in wide use in the food industry or the companies were ready to implement them.
523
Information on the use of different microbiological methods in the food industry can also be obtained from
524
market reports. Recent reports reveal similarities and differences in food safety testing geographically. In
525
15 years the total test volumes have increased by 128 per cent, with pathogen testing taking a bigger
526
portion of the total volume (from 13.7% to 23.2%) (Weschler, 2013). In the US 78% of the tests are for
527
routine and 22% for specific pathogens, while in the EU the figures are 82% and 18%, respectively
528
(Weschler, 2012). The organism types (Total Viable Organisms (TVO), Coliforms, Yeast/Mould,
529
Staphylococci), tested routinely are similar in both the USA and EU but the tested specific pathogens differ:
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monocytogenes is tested much more often (34% in EU vs. 3% in USA), with E. coli tests amounting to just
532
1.8%. Salmonella is tested equally in both regions.
533
Raw material samples account for 26% of all food samples collected (in the USA 8%, EU 16%), in-
534
process/environmental samples for 25% (USA 44%, EU 26%), and end products for 49% (USA 48%, EU 59%),
535
so there is significant variation geographically (Weschler, 2013). This reflects the different trends and
536
perceptions about food safety.
537
The used detection methods also vary according to geographical location. In the USA, mostly convenience
538
methods are used for routine testing (66% in USA vs. 23% in EU), whereas in the EU traditional tests prevail
539
(65% in EU vs. 20% in USA) (Weschler, 2012). In pathogen testing, the USA uses antibody- and molecular-
540
based methods (together 88%), while the EU relies on traditional or convenience culturing (66%).
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5. Future trends
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In addition to attempts to improve or modify the existing food pathogen detection methods, new
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technologies are being developed. For example a proof-of-concept study reported by Chai, Horikawa, Li,
546
Wikle, and Chin (2013) introduced a phage-coated magnetoelastic biosensor for detecting Salmonella
547
bacteria on fresh food surfaces in real time and in-situ. The DL of the system was < 1.5 x 103 CFU/mm2.
548
Another trend seen in the literature is the simultaneous targeting of multiple pathogens during or in one
549
assay, be it immunological or based on some form of amplification or sensor method. L. monocytogenes is
550
frequently being identified together with other food pathogens, especially Salmonella and E. coli.
551
Methods based on isothermal amplification, e.g. LAMP, are increasing in the literature (Niessen et al.,
552
2013), also for L. monocytogenes, and detection kits relying on the same technology are emerging in the
553
market (Table 2). According to Niessen et al. (2013) food safety and quality laboratories in the public sector
554
and industry are already using LAMP or planning to use it.
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556
patenting statistics. Therefore, we carried out a worldwide survey on the Espacenet database for the
557
detection, identification or determination of Listeria, which yielded about 120 references from the years
558
2000 to 2014. Out of these, 65% are from the Far East (44% from China alone) reflecting the current trend
559
in assigning countries. The USA is the next with 15%, followed by Korea (10%), Japan (8%) and France (6%).
560
Most applications/patents were based on PCR or other amplification technologies, followed by
561
immunoassays and other types of methods. Amplification-based methods were also the most numerous
562
category in patent publications from Europe and the USA, but the second place was held by inventions
563
concerning the culture of Listeria.
564
Nucleic acid-based Listeria detection methods account for a much bigger portion of patents than of
565
currently validated alternative test kits (70% vs. 43%). Likewise, the non-validated alternative methods are
566
mostly nucleic acid amplification or probing methods (Table 2). It is therefore anticipated that the list of
567
validated alternative methods will also be dominated by nucleic acid methods in the near future.
568
Based on their recent reports, Strategic Consulting, Inc. (SCI) (SCI, 2014) suggests some current trends in
569
food safety testing. First, food testing will increase because of public concern, active media, and increasing
570
regulations, but the use of rapid methods will grow at a different pace in different areas. Second, food
571
safety testing is shifting to third-party laboratories due to the growing amount of effort and investments
572
needed to carry out the analyses in-house, in addition to the expectations of accreditation. Third, more
573
environmental testing, especially for pathogens, will be carried out at food production plants because of
574
new regulations.
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6. Conclusions
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There exists a wide selection of methods for targeting L. monocytogenes and Listeria spp. that can be
579
concluded in 48 hours. The number of validated methods is close to one hundred, and additionally tens of
580
other commercial methods are on the market. The alternative methods in the recent literature rely on
24
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582
of the standard culture methods have also been achieved by different alternative technologies but in a
583
shorter detection time. Particularly RT-PCR-based detection of authentic food samples is rivalling the
584
standard culture methods in terms of the detection limit. In addition, the enrichment time is shortening or
585
enrichment is not included in new methods. Especially the different sensor-based approaches with an
586
analysis time of a few hours favour direct detection, but in general their DLs do not yet quite match those
587
of amplification methods. Some of the rapid tests in the literature have been thoroughly validated with
588
reference strains and different food matrices, but mainly they are more or less demonstrative and need
589
further evaluation. The users of the methods, laboratories in the industry and the public sector, are slowly
590
but steadily adopting rapid methods for use along with the standard ones or even replacing them.
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7. Acknowledgements
593
This work was supported by the project A31588 (Food Safety Cluster) funded by the Council of Oulu Region
594
from the European Regional Development Fund (ERDF) of the European Union.
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References Alessandria, V., Rantsiou, K., Dolci, P., & Cocolin, L. (2010). Molecular methods to assess Listeria
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monocytogenes route of contamination in a dairy processing plant. International Journal of Food
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Microbiology, 141, S156-S162. Amagliani, G., Giammarini, C., Omiccioli, E., Brandi, G., & Magnani, M. (2007). Detection of Listeria
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monocytogenes using a commercial PCR kit and different DNA extraction methods. Food Control,
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18(9), 1137-1142.
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Todd, E. C. D., & Notermans, S. (2011). Surveillance of listeriosis and its causative pathogen, Listeria monocytogenes. Food Control, 22, 1484-1490. Vázquez-Boland, J. A., Kuhn, M., Berche, P., Chakraborty, T., Domínguez-Bernal, G., Goebel, W., ...Kreft, J.
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paramagnetic beads coated with recombinant Listeria phage endolysin-derived cell-wall-binding
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domain proteins for separation of Listeria monocytogenes from raw milk in combination with culture-
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amplification (lamp) method for detecting Listeria monocytogenes in raw milk. Journal of Food
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Wang, H., Li, Y., Wang, A., & Slavik, M. (2011). Rapid, sensitive, and simultaneous detection of three
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LWT - Food Science and Technology, 44, 351-362.
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1 Table 1 Detection limits of the standard and some alternative, rapid Listeria monocytogenes (LM) identification methods Detection limit
Matrix
Enrichment | Total time if given
ISO 11290 FDA USDA-FSIS
<1 CFU/25 g <1 CFU/ml <1 CFU/g in 25 g
all foods dairy, fruit & vegetable, seafood meat, poultry, egg, environmental
96/120 h|4–7 d 76/100 h|4–7 d 66–74/90–98 h|4– d
RNA RT-PCR IMS+RT-PCR Multiplex-RT-PCR
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Reference
ISO, 2004a,b Hitchins & Jinneman, 2013 FSIS, 2013
6 h|2 d
5/2/9
Kretzer et al., 2007
14 h|15 h
11/5/13
Shim et al., 2008
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lettuce, cheese, fish, meat, milk (A/N 275 ) meat (A 15/N 116)
10 CFU/ml 20 CFU/ml 5–10 CFU/g <5 CFU/ml 20–50 CFU/ml 2 >10 CFU/g
2
milk (A) cheese, fish, milk, strawberries, swab (N 22) meat, seafood, dairy products (A 30/N 190) milk (A) meat, lettuce (A) vegetables (N 191)
No|2 h 11 h|16 h 24 h| 2 h at RT|4 h No|2 h 7 h|
1/-/3 1/-/2/4/38 1/-/2 1/-/2 2/-/-
Cho & Irudayaraj, 2013 Jaakohuhta et al., 2007 Portanti et al., 2011 Cho I. et al.,2014 Wang et al., 2011 Moreno et al., 2012
1 CFU/g 8 CFU/ml c 1.45 CFU/ml 10 CFU/25 g 1 CFU/ml or g 1 CFU/15 g 2–4 CFU/25 g 1 CFU/25 g 1–5 CFU/25 g
sausage, cheese (A) cheese, meat(A) milk, sewage water, vessel surfaces (A/N 27) liquid egg (A) milk, meat (A) cheese, pâté (A/N 76) meat, dairy products, vegetables (A) meat, fish, cheese, dairy products (A 61/N 144) meat, fish, dairy products, desserts (A 16/N 175) yogurt (A) meat (A) fish (A) milk (A)
24 h|2 d 5+17 h| 6 h| 15 h| 8 h|18 h 24 h| 24 h|27 h 24+6 h|2 d 24+4 h|2 d
1/-/1/-/5/6/2 1/-/2 15/8/262 100/30/29 d 9/-/d 1/-/d ?/-/-
Amagliani et al., 2006 Rip & Gouws, 2009 Zeng et al., 2006 Germini et al., 2009 Chiang et al., 2012 Rossmanith et al., 2006 Rodriquez-Lazaro et al., 2014 Oravcová et al., 2007 O’Grady et al., 2009
No| No| No|3.5 h 18±2 h|2 d
1/-/1/1/40 d 2/-/33/7/50
D’Urso et al., 2009 Ye et al., 2012 Duodu et al., 2009 Omiccioli et al., 2009
10 CFU/10 g 1 CFU/ml 10–40 CFU/g 1 CFU/5x25 ml
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10 CFU/10 g
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10 CFU/g
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Culture Phage-IMS Immunoassays IMS + Immunochromatography IMS + Lateral flow Time resolved FIA ELISA IMS + IA Multiplex-IMS + IA FISH Amplification PCR
Specificity tested LM/ Listeria spp./ other strains
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Method
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2 24 h|< 30 d 18 h|24 h No|2.5 h 24 h| No|
11/11/58 2/4/67 45/5/10 23/8/8 2/-/12
Ruiz-Rueda et al., 2011 Köppel et al., 2013 Wan et al., 2012 Cho A.R. et al., 2014 Wang, et al., 2010
blueberries (A) tomato extract (A) milk (A) meat (A)
No| No| No|1 h 18 h|24 h
3/-/4 1/-/1 ?/-/1/5/5
Davis et al., 2013 Radhakrishnan et al., 2013 Sharma & Mutharasan, 2013b Ohk et al., 2010
b
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Biosensors 2 Amperometric 10 CFU/g Impedance spectroscopy 4 CFU/ml 2 3 Piezoelectric 10 –10 CFU/ml 2 Fibre-optic 10 CFU/25 g a 0.1 CFU/g after 24 h enrichment
dairy products, fish, vegetables, meat (A/N 54) cheese, yogurt (A 250/N 212) meat, milk powder (A 4) milk (A) milk (A/N 125)
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5 CFU/25 g 2 CFU/g 3 10 CFU/ml e 2 CFU/ml 186 CFU/ml
A = tested artificially contaminated samples; N = tested naturally contaminated samples
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ACCEPTED MANUSCRIPT Table 2 Some commercially available non-validated L. monocytogenes and Listeria spp. tests Test type
Technical Service Consultants Ltd Creative Diagnostics AdipoGen
Color
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HybriScan Listeria monocytogenes Listeria spp. tests Compact Dry LS Hygiena InSite™ Rapid Environmental Listeria Test Listeria Isolation Transwab
Path-Chek Hygiene Pathogen System Listeria Latex Agglutination Test Listeria Rapid Test Kit RIDASCREEN Listeria Singlepath® Listeria Listeria Detection Listeria Listeria spp. and Listeria monocytogenes Multiplex PCR with internal Control innuDETECT Listeria spp. Assay rapidSTRIPE Listeria Assay
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Immunochromatographic ELISA Immunochromatographic RT-PCR RT-PCR RT-PCR RT-PCR RT-PCR
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Merck ingenetix GmbH Primerdesign Microbial SL BioGX GEN-IAL GmbH
Liferiver/BioSB NORGEN Biotek Corp. Diatheva Diatheva Q-Bioanalytic GmbH
RT-PCR PCR/RT-PCR PCR RT-PCR PCR/RT-PCR
R-Biopharm AG
RT-PCR
SA Scientific Eiken Chemical CO. Ltd. / Mast Group Ltd. 3M
LAMP, Real time LAMP, Real time
TwistDx
Sigma Aldrich
IAMP (Recombinase) Realtime Sandwich hybridization
R-Biopharm AG Hygiena
Color Color
Medical Wire & Equipment Microgen Bioproducts Creative Diagnostics Hardy Diagnostics R-Biopharm AG Merck IDLabs BioGX GEN-IAL GmbH
Color
Analytik Jena Analytik Jena
RT-PCR PCR & lateral flow
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Listeria Monocytogenes Rapid Test Listeria monocytogenes SURE mono ELISA Kit Singlepath® L’mono BactoReal Listeria monocytogenes genesig Listeria monocytogenes Listerfast Listeria monocytogenes Listeria monocytogenes with internal Control Listeria monocytogenes Real Time PCR Kit Listeria Monocytogenes PCR Detection Kit Listeria monocytogenes PCR detection Kit Listeria monocytogenes FLUO kit QuickBlue (RealQuick) Listeria monocytogenes SureFood® PATHOGEN Listeria monocytogenes PLUS Listeria monocytogenes Detection Kit Loopamp Listeria monocytogens DetectionKit Molecular Detection Assay Listeria monocytogenes TwistAmp® exo+ListeriaM
Manufacturer/Supplier
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Test Listeria monocytogenes tests SwabSURE Listeria P (also L. ivanovii)
LAMP, Real time
Color Latex Agglutination Immunoassay Immunoassay Immunochromatographic PCR RT-PCR RT-PCR
2
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PCR/RT-PCR RT-PCR Probe & fluorescence microscopy
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QuickBlue (RealQuick) Listeria spp. SureFood® BAC Listeria Screening PLUS VIT® Listeria
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Highlights: • overview of currently available, rapid (<48 h) L. monocytogenes detection methods • focus on naturally or artificially contaminated food and environmental samples • summary of the most rapid and sensitive methods • many methods as sensitive as standard methods, but much faster
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S0956-7135(15)00130-9
DOI:
10.1016/j.foodcont.2015.02.037
Reference:
JFCO 4328
To appear in:
Food Control
Received Date: 16 September 2014 Revised Date:
26 January 2015
Accepted Date: 3 February 2015
Please cite this article as: Välimaa A.-L., Tilsala-Timisjärvi A. & Virtanen E., Rapid detection and identification methods for Listeria monocytogenes in the food chain – a review, Food Control (2015), doi: 10.1016/j.foodcont.2015.02.037. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Rapid detection and identification methods for Listeria monocytogenes in the food
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chain – a review
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Anna-Liisa Välimaaa,1, Anu Tilsala-Timisjärvib,1 and Elina Virtanenb
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a
New business opportunities, National Resources Institute Finland (LUKE), Oulu, Finland
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b
Green technology, National Resources Institute Finland (LUKE), Oulu, Finland
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These authors contributed equally to this work.
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Anna-Liisa Välimaa
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National Resources Institute Finland (LUKE)
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P.O. Box 413
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FI-90014 University of Oulu
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Finland
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email: [email protected]
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phone: +358-40 195 8286
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Abstract
20 Listeria monocytogenes is a facultative pathogenic saprophyte. It can cause a severe disease, listeriosis,
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which is currently considered to be one of the leading food-borne diseases worldwide. L. monocytogenes
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can be found in raw and processed foods. Particularly ready-to-eat (RTE) foods are sources of Listeria
24
infections. RTE foods have a long shelf life, because they are stored at low temperatures and in vacuum or
25
modified atmosphere packages. Additionally, they are usually consumed without any additional cooking. As
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L. monocytogenes can multiply over a wide range of pH and osmolarity, at low temperatures, and both
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under aerobic and anaerobic conditions, this is a particular concern and necessitates control along the food
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chain. A wide variety of culture and alternative methods have been developed in order to detect or
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quantify this pathogen in food. Here are presented the most rapid and sensitive methods (< 48 h) found in
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the literature that have been used with artificially and/or naturally contaminated food samples. In addition
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to being much more rapid, many of them were as sensitive as the standard methods. However, many
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methods still need to be more thoroughly validated.
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Keywords: Listeria monocytogenes, food, detection, rapid, alternative, methods
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1. Introduction
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Listeria monocytogenes is considered a human pathogen. It has been isolated from urban and natural
39
environments, animals and humans (Orsi, den Bakker, & Wiedmann, 2011), and food and food processing
40
plants including abattoirs and smokehouses (Moretro & Langsrud, 2004).
41
L. monocytogenes can grow over a temperature range from -0.4 °C to 45 °C (Junttila, Niemelä, & Hirn, 1988;
42
Walker, Archer, & Banks, 1990), over a pH range from 4.0 to 9.6 (optimum 6–8) (Farber & Peterkin, 1991),
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at water activity (aw) levels of even 0.90 (Farber, Coates & Daley, 1992), and both under aerobic and
44
anaerobic conditions. Moreover, L. monocytogenes is able to adhere to a variety of food contact surfaces,
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processing facilities for several months or even years (Miettinen, Björkroth, & Korkeala, 1999; Norton et al.,
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2001; Orsi et al., 2008) as biofilms (Pereira da Silva & Pereira da Martinis, 2013). Protected in biofilms it can
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tolerate high concentrations of many environmental agents, such as sanitizers, disinfectants, and
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antimicrobials, which may result in contamination of food contact surfaces (Carpentier & Cerf, 2011).
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L. monocytogenes may occur in raw foods and in processed foods that are contaminated during and/or
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after processing (EFSA, 2013a). Particularly ready-to-eat (RTE) foods, such as fishery products, heat-treated
52
meat products, and cheese, are often sources of Listeria infections. RTE foods can have a long shelf life.
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They are stored at low temperatures and in vacuum or modified atmosphere packages, and are usually
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consumed without any additional cooking. L. monocytogenes has also been found in plant food, e.g. salted
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mushrooms, broccoli, coleslaw, and cantaloupe (EFSA, 2013b). Its ability to grow at low temperatures and
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both under aerobic and anaerobic conditions and in modified atmosphere packaging (Swaminathan &
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Gerner-Smidt, 2007) makes it a great concern for the food industry and necessitates control along the food
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chain (Lambertz, Ivarsson, Lopez-Valladares, Sidstedt, & Lindqvist, 2013) in order to prevent listeriosis, a
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severe threat to public health.
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The first well-documented outbreak of foodborne listeriosis was reported in Canada in 1983, with coleslaw
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as the implicated food (Schlech et al., 1983). Since then, several other outbreaks have been reported. One
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of the deadliest occurred in 2011 in the United States of America (USA); it was associated with cantaloupe
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and caused 146 invasive illnesses, one miscarriage and 30 deaths (Laksanalamai et al., 2012). The risk
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groups for listeriosis are pregnant women, infants, the elderly, and people with weak immune systems;
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among them, it may cause abortion or stillbirth, sepsis, pneumonia or meningitis and serious infections of
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the nervous system, depending on the risk group (Todd & Notermans, 2011).
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L. monocytogenes can access the human food chain directly or through farm animals (zoonotic disease)
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(Gahan & Hill, 2014). The infective dose is suspected to be high; contamination levels in food responsible
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for listeriosis cases are typically >104 CFU/g (Ooi & Lorber, 2005; Vázquez-Boland et al., 2001). Consuming
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foods that contain low levels (<102 CFU/g) of L. monocytogenes is unlikely to cause clinical disease (Chen,
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immunocompromised population (McLauchlin, Mitchell, Smerdon, & Jewell, 2004; Vázquez-Boland et al.,
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2001).
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The case-fatality rate of listeriosis can be high, 20–30% (Todd & Notermans, 2011). In the EU, 1476
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confirmed cases were reported in 2011. Of all the zoonotic diseases under EU surveillance, listeriosis
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caused the most severe human disease with a fatality rate of 12.7% (EFSA, 2013c).
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In the USA, 1600 listeriosis cases are estimated yearly, with 250 deaths (Scallan et al., 2011). The total
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economic cost of listeriosis, comprising health care costs, lost productivity, and diminished quality of life, is
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estimated to amount to almost USD 2.04x1012 and the cost per case to nearly USD 1,282,000 annually
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(Byrd-Bredbenner, Berning, Martin-Biggers, & Quick, 2013). Sales reductions of RTE foods due to L.
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monocytogenes recalls were estimated to be 22–27% after the event (Thomsen, Shiptsova, & Hamn, 2006).
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Legislation addressing L. monocytogenes contamination in food varies. In the EU, for a healthy human
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population, foods not exceeding the limit of 100 CFU/g are considered safe (EC, 2005). If the foods are able
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to support L. monocytogenes growth and the food processor cannot demonstrate that this limit is not
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exceeded during the shelf life, L. monocytogenes must be absent. In any case, in RTE products intended for
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infants and for special medical purposes, L. monocytogenes must be absent in 25 g. In the USA, the Food
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and Drug Administration (FDA) has a zero tolerance policy for L. monocytogenes in RTE products (FDA,
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2003); that said, in a nonbinding compliance policy guide, the L. monocytogenes level was set at <100
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CFU/g for RTE foods that do not support its growth (FDA, 2008).
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Here, rapid methods used for the detection of L. monocytogenes in food are presented, especially in the
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context of the production environment and finished products. The recently published literature describing
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L. monocytogenes detection, quantification or identification in naturally or artificially contaminated food
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samples is reviewed. Market report information and other data on the usage of microbiological detection
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methods in the food industry are included, focusing on Europe and the USA. Patent information is also
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covered. The aim is to give an overview of selected current rapid (<48 h) L. monocytogenes detection or
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identification methods and bring them to the awareness of all interest groups in the food production field
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who are involved in ensuring food safety.
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2. Detection and quantification methods for L. monocytogenes and Listeria spp. in food
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Various microbiological methods have been used for decades in the food industry for the detection of
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bacteria. Legislation in the EU and USA still relies on methods based on culture assays. These methods are
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designed to enable the detection of a single target cell in a sample, and this is also the desired
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characteristic of any rapid method intended to replace a culture-based one (Dwivedi & Jaykus, 2011). As
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“rapid” may mean a shorter time, but can also refer to better flow-through or handling of multiple samples
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for greater convenience and automation of work, Jasson, Jacxsens, Luning, Rajkovic, and Uyttendaele
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(2010) suggested using the term “alternative method”. Wiedmann, Wang, Post, and Nightingale (2014)
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have thoroughly outlined criteria for evaluation of rapid detection methods in the food industry, which may
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help in the selection of a suitable one. Microbiological methods can be classified as quantitative or
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qualitative. Quantitative methods (enumeration) aim at determining the number of bacteria present,
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directly or indirectly, in a given sample. Qualitative methods will demonstrate whether the target
112
bacterium is present or not in the sample.
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If a food sample also contains other Listeria species, particularly L. innocua, L. monocytogenes may be
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overgrown by them, which may lead to false negatives (Gnanou Besse et al., 2010). However, when Listeria
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spp. is targeted, a positive result may also indicate presence of L. monocytogenes.
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2.1. Classical cultural methods
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Classical detection methods employing enrichment-plating techniques are used for the testing of presence
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or absence of pathogens, usually in 25 g of food, and the overall detection limit (DL) is ca. 1–5 CFU/test
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portion (Jasson et al., 2010).
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salts, preservatives, and other chemicals or natural antimicrobial compounds (Wu, 2008). Additionally,
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viable bacteria may respond to various environmental stresses by entering a dormancy state where the
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cells remain viable, but non-culturable (VBNC) on standard laboratory media (Oliver, 2010). If resuscitated
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from this VBNC state, the cells can again be cultured and cause infection. Therefore, their recovery during
126
culturing procedures is critical.
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Due to these sub-lethally injured and VBNC states, the detection method is often divided into a two-step
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enrichment process. The pre-enrichment step in a non- or half-selective medium resuscitates the injured
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target organisms and increases them. It also dilutes inhibiting compounds and rehydrates bacterial cells
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derived from dried or processed food matrices (Dwiwedi & Jaykus, 2011). The second enrichment step in a
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selective medium (containing e.g. different salts and antibiotics) suppresses the background flora and
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increases the target pathogen, resulting in a million-fold multiplication of the target, enabling its isolation
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or detection (Jasson et al., 2010). Presumptive positive colonies are isolated on a selective differential agar
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medium. If typical colonies are absent, the analysis is completed. The presumptive colonies are confirmed
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by additional morphological, biochemical, physiological, and/or serological testing. It can take up to 4 days
136
to detect the pathogen presumptively (appearance of typical colonies on the medium) or to get a negative
137
result (no typical colonies), and confirmation of the positive result can take around one week (Dwiwedi &
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Jaykus, 2011; Jasson et al., 2010).
139
Various official culture-based L. monocytogenes detection methods (e.g. International Organization for
140
Standardization (ISO) and FDA), and their differences have been outlined by Zunabovic, Domig, and Kneifel
141
(2011). They are recommended for or have been established with different target matrices, and they use
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different pre-enrichment media and incubation times, whereas the incubation temperature is the same (30
143
°C). The selective enrichment media, incubation temperature (30/35/37 °C), and time vary according to the
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standard.
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Convenience methods are conventional microbial analysis methods that have been modified or automated
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to make them faster and less laborious, e.g. using fluorogenic or chromogenic substrate in selective media
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and avoiding the need for sub-culturing and further biochemical testing (Mandal, Biswas, Choi, & Pal,
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2011). Commercial morphological, biochemical, and physiological tests are also available for identifying
149
pathogens using conventional methods.
150 2.2. Rapid or alternative detection methods
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The (alternative) diagnostic methods often combine various technologies (culture, immunoassays, nucleic
153
acids). Table 1 presents the detection limits and enrichment times of the standard and some alternative
154
methods for Listeria monocytogenes determination in food. The limit for ISO 11290 is based on various
155
validation certificates or reports available at the AFNOR NF Validation website (http://nf-
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validation.afnor.org/en/alternative methods/). The rapid methods (≤ 48 h) found in the literature are
157
included here if their reported detection limit is ≤104 cells and they have been tested in food. The DL can be
158
given as the limit in the food sample before enrichment or as the limit for the molecular test (Wiedmann et
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al., 2014). Thus, because the DLs of the methods are presented in various meanings or units in the
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literature, it is not possible to make direct comparisons.
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2.2.1. Immunoassays
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Immunological assays are based on the specific binding of an antibody and an antigen. The capturing part
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of the antigen recognizes the epitopes on the antibody and binds to them. A monoclonal antibody may
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identify a particular epitope whereas a polyclonal antibody may recognize several epitopes of a certain
166
antigen or many antigens. The DL is influenced by the antibody and the immunological assay type (about
167
103–107 CFU/ml in the literature). In order to achieve the set detection limit criterion, enrichment is usually
168
needed. The average sensitivity of immunological methods has been improved in the examples given here.
169
The most widely used immunological methods in food diagnostics are lateral flow immunoassay, enzyme-
170
linked immunosorbent assay (ELISA), enzyme-linked fluorescent assay (ELFA), and immunomagnetic
171
separation (IMS). IMS is included in many detection methods for separating and concentrating the target
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sample matrix.
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In lateral flow immunoassay or immunochromatography the reaction between an antibody and its target is
175
detected visually. It relies on the migration of the target in a sample to the antibody that is immobilized on
176
a membrane surface. A lateral flow chromatographic enzyme immunoassay together with a magnetic
177
concentration step was demonstrated to detect 102 CFU/ml L. monocytogenes in spiked milk within two
178
hours (Cho & Irudayaraj, 2013) with monoclonal L. monocytogenes-specific antibodies LZF7 and LZH1.
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Blažková, Koets, Rauch, and van Amerongen (2009) combined polymerase chain reaction (PCR) and lateral
180
flow immunoassay for detecting the amplified products for Listeria spp. and L. monocytogenes. An aliquot
181
of the PCR products was added to an assay device, and the presence of a specific amplicon was visualized
182
with a dark line. It detected <10 cells/25 ml milk within 28 hours including 24 h enrichment, and was tested
183
with 24 authentic food samples and found to be in concordance with the ISO standard method. Kovačević
184
et al. (2009) evaluated commercial assays for identifying Listeria spp. at meat processing facilities with
185
environmental samples from various production stages. A lateral flow assay was found to be as specific as
186
culturing and a PCR assay and not significantly less sensitive with >300 tested samples.
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In ELISA an immunological and an enzyme assay are combined, usually in a sandwich ELISA form. Shim et al.
188
(2008) developed an immunochromatography (ICG) strip test and an ELISA test with IMS (3B12-17 MAb) for
189
qualitative detection of Listeria spp. and L. monocytogenes. They were used with 116 naturally
190
contaminated meat samples, and ICG-IMS could detect these pathogens in 15 hours, with a limit of 102
191
CFU/10 g. An ELISA test targeting L. monocytogenes in meat, seafood, and dairy products was validated
192
against the ISO method by Portanti et al. (2011) using 190 naturally and 30 artificially contaminated
193
samples. It used a single enrichment step and produced results in concordance with the standard. The DL
194
was 6.6 x 103 CFU/ml and the relative DL 5–10 CFU/g in spiked samples.
195
In order to enhance the sensitivity of ELISA, a fluorescent label can be added to the antibodies (ELFA). A
196
time-resolved fluorescent immunoassay for Listeria spp. (Jaakohuhta, Harma, Tuomola, & Lovgren, 2007)
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was compared to a commercial ELFA test by measuring 22 naturally contaminated cheese, fish, milk,
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and was more sensitive (20 CFU/ml). A commercial ELFA, a commercial RT-PCR designed for food, and
200
culturing were evaluated for detecting L. monocytogenes in 50 vacuum-packed meat products (Netschajew,
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Fredriksson-Ahomaa, Sperner, & Stolle, 2009). Immunoassay and culture found only some positives, while
202
the RT-PCR detected 32 as positive after an overnight enrichment. The difference was explained by a lower
203
contamination level or high number of unculturable cells.
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Different types of fluorescent systems have been developed for increasing the sensitivity of immunoassays.
205
L. monocytogenes was targeted simultaneously with E. coli and S. typhimurium using magnetic nanobeads
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with polyclonal antibodies for separation and quantum dot (QD) fluorescence for detection in 2 hours
207
(Wang, Li, Wang, & Slavik, 2011). A DL of 20–50 CFU/ml in inoculated ground beef, chicken, broccoli, and
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lettuce samples was reported. Cho, Mauer, and Irudayaraj (2014) developed a slightly more sensitive
209
method applying immunomagnetic beads with L. monocytogenes-specific monoclonal antibodies for
210
capture and antibody-BSA labelled with fluorophores for detection. The feasibility of the L. monocytogenes
211
system was demonstrated with spiked milk in 4 hours (including a 2 h incubation at RT), detecting <5
212
CFU/ml of L. monocytogenes. A multiplex detection with E. coli and S. typhimurium (mixed culture samples)
213
was also demonstrated. Other Listeria species were not included for testing in either of the above multiplex
214
methods.
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2.2.2. Biosensors
216
A biosensor is an analytical device converting a biological response into an electrical signal. It consists of a
217
bioreceptor (microorganism, cell, enzyme, antibody, nucleic acid) and a transducer (Velusamy, Arshak,
218
Korostynska, Oliwa, & Adley, 2010). The transducer may be optical (Raman/Fourier transform IR-
219
spectroscopy, surface-plasmon resonance, optic fibres), electrochemical (amperometric, impedimetric,
220
potentiometric, conductometric), mass-based (piezoelectric, magnetic) or thermal. Biosensors have been
221
recently reviewed in the context of food pathogens (e.g. Arora, Sindhu, Dilbaghi, & Chaudhury, 2011; Arora,
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Sindhu, Kaur, Dilbaghi, & Chaudhury 2013; Sharma & Mutharasan, 2013a). Truly rapid biosensor methods
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Rogers, and Suni (2013), who used impedance spectroscopy and monoclonal IgG1 antibodies, and achieved
225
a calculated DL of 4 CFU/ml for L. monocytogenes in an artificially inoculated tomato extract without
226
enrichment.
227
Davis et al. (2013) used screen-printed carbon electrode (SPCE) strips as disposable amperometric sensors
228
for ELISA with commercial anti-L. monocytogenes antibodies and demonstrated detection of 102 CFU/g L.
229
monocytogenes from spiked blueberries in about one hour. Instead of an electrochemical transducer,
230
Sharma and Mutharasan (2013b) demonstrated L. monocytogenes detection in milk using a mass-based
231
piezoelectric cantilever biosensor and a commercial polyclonal anti-L. monocytogenes antibody, with a limit
232
of 103 cells/ml. Adding a third antibody binding step into the method enabled the detection of 102 cells/ml
233
in one hour.
234
A fibre-optic immunosensor system based on sandwich immunoassay with MAb C11E9 and fluorescence
235
detection was developed by Geng, Morgan, and Bhunia (2004). They demonstrated that this system
236
detected L. monocytogenes in artificially or naturally contaminated hot dogs and bolognas, in 24 hours
237
after 20 h enrichment (10–103 cells/g inoculations). An oligonucleotide aptamer specific for internalin A
238
was used as a reporter molecule in a fibre-optic biosensor (Ohk, Koo, Sen, Yamamoto, & Bhunia, 2010). The
239
DL of the assay was 103 CFU/ml, and it could detect 4 CFU/g spiked L. monocytogenes in deli meats (beef,
240
chicken, and turkey) after 18 h enrichment. The type of enrichment broth used influenced the success of
241
the detection. A DL of 3 x 102 CFU/ml was obtained for L. monocytogenes by a fibre-optic sensor together
242
with immunomagnetic cell separation using anti-InlA MAb-2D12 antibodies (also specific for L. ivanovii)
243
(Mendonҫa et al., 2012). In soft cheese and hotdogs 10–40 CFU/g were detected after 18 h enrichment.
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The total analysis time of these example methods is < 24 h or only a few hours, but in general their DLs are
245
higher than those of amplification methods, so their sensitivity needs improvement. Some methods also
246
need further validation with reference species and different types of food samples.
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2.2.3. Bacteriophage-based detection methods
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ACCEPTED MANUSCRIPT Bacteriophage-based applications in food safety and diagnostics have been reviewed earlier (Lu, Bowers, &
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Koeris, 2013; Singh, Poshtiban, & Evoy, 2013; Smartt et al., 2012). The mentioned advantages of phage-
251
based detection include target specificity, sensitivity, distinction of live and dead cells, and ease of detector
252
amplification. Commercial immunoassays using bacteriophage tail proteins for detecting Listeria and L.
253
monocytogenes are available. A system utilizing phages to produce illuminative reporter proteins in Listeria
254
cells is also on the market. In the literature, phage-based proteins have been applied for the separation and
255
detection of Listeria through the use of paramagnetic beads and culturing (Kretzer et al., 2007). The
256
bacteria were detected from spiked and 275 naturally contaminated food samples (lettuce, cheese, salmon,
257
meat, milk). A limit of 102 CFU/g was obtained in all food materials after 6 h pre-enrichment and a limit of
258
0.1 CFU/g in all categories except soft cheese after 24 h enrichment. The sensitivity was better than with
259
the standard culture method. Use of these phage protein-coated beads for separating L. monocytogenes
260
directly from artificially contaminated raw milk samples and detecting them by plating or RT-PCR was
261
evaluated by Walcher et al. (2010). The achieved DL by IMS, DNA-isolation, and RT-PCR (on prfA;
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transcriptional activator of the virulence factor) was 102–103 CFU/ml.
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2.2.4. Fluorescent in situ hybridization (FISH)
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Ribosomal RNA targeting probes are commonly used with FISH, where the hybridization is detected by
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fluorescence microscopy. FISH in the context of food diagnostics has been recently reviewed by Rohde,
267
Hammerl, Appel, Dieckmann, and Al Dahouk (2015). Detection of viable Listeria spp. (23S rRNA probe) or L.
268
monocytogenes (16S rRNA probe) cells from spiked smoked salmon, camembert and mozzarella cheese,
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ham, and cabbage samples (102–3x103 CFU/ml) has been carried out using FISH with filter cultivation
270
(Fuchizawa, Shimizu, Kawai, & Yamazaki, 2008; Fuchizawa, Shimizu, Ootsubo, Kawai, & Yamazaki, 2009).
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The hybridization was observed by fluorescent microscopy. The method was equal to a plating method in
272
enumerating the cells but took less time (enrichment and FISH 12–16 h in total). Viable L. monocytogenes
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were detected from 191 fresh and frozen vegetable samples by culture (ISO Standard), PCR targeting 16S
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rDNA and hlyA for Listeria genus and L. monocytogenes, and Direct Viable Count (DVC)-FISH targeting L.
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monocytogenes 16S rRNA (Moreno et al., 2012). In DVC bacteria are grown in a medium that prevents cell
276
division and promotes elongation of cells with metabolic activity, which are counted as viable. With culture,
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PCR, or DVC-FISH 4%, 10%, or 33% of the samples were positive, respectively. DVC-FISH enabled the
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detection of the viable cells (7.4x102–9.4x104 CFU/g) after 7 h culturing.
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In the literature, various amplification methods are the most widely used alternative methods for L.
282
monocytogenes detection in food samples. Quantitative PCR (qPCR) or real-time PCR (RT-PCR) application
283
in food microbiology and diagnostics has been reviewed earlier (Cocolin, Rajkovic, Rantsiou, & Uyttendaele,
284
2011; Postollec, Falentin, Pavan, Combrisson, & Sohier, 2011), likewise the use of multiplex-PCR for
285
targeting food microbes (Settanni & Corsetti, 2007). Other amplification methods have emerged, e.g.
286
isothermal amplification (including loop-mediated isothermal amplification (LAMP) and hyperbranching
287
rolling circle amplification (HRCA)), and nucleic acid sequence-based amplification (NASBA) for amplifying
288
RNA templates. An overview of current technologies for isothermal amplification has been presented by
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Craw and Balachandran (2012.)
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2.2.5.1. Conventional PCR
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Conventional qualitative or semi-quantitative PCR relies on the endpoint analysis of the amplified products
293
(electrophoresis, fluorescence). A commercial, non-validated Listeria monocytogenes detection kit was
294
evaluated on spiked, enriched (24 h) raw pork sausage and mozzarella cheeses (Amagliani, Giammarini,
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Omiccioli, Brandi, & Magnani, 2007), and it detected 1 CFU/g L. monocytogenes. While optimizing an
296
internal control (IAC) for a L. monocytogenes PCR-assay, Rip and Gouws (2009) detected 8 CFU/ml from
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spiked and enriched (22 h) camembert cheese and ostrich meat samples targeting the hly gene. Delibato et
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al. (2009) have described a combined PCR and microfluidic chip-based automated electrophoresis system
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for detecting L. monocytogenes in 50 naturally contaminated food samples (RTE meat, cheese, smoked
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of positive samples.
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Multiplex PCR systems enable the detection of different species at the same time in one run or different
303
targets in one reaction. A system for identifying viable Listeria genus and different Listeria species (L.
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monocytogenes, L. innocua, and L. grayi) was developed using reverse transcription-based PCR targeting
305
iap mRNA (Rattanachaikunsopon & Phumkhachorn, 2012). The DL was 50 CFU/ml in pure cultures. The
306
feasibility of the system was evaluated with spiked meat samples (102 CFU/ml) with one-hour enrichment.
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Zeng, Zhang, Sun, and Fang (2006) used multiplex-PCR for detecting Listeria spp. and L. monocytogenes iap
308
and hly genes in samples from spiked milk and isolates from a milk processing environment (raw milk,
309
sewage water, vessel surfaces) to contain L. monocytogenes. PCR and API identified the same isolates. The
310
DL of the PCR was 1.4 x 102 CFU/ml directly in spiked milk but an enrichment of 3–6 h yielded a limit of 1.45
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CFU/ml. Multiplex-PCR for L. monocytogenes (prfA gene), Salmonella spp., and E. coli was demonstrated in
312
liquid egg samples (Germini, Masola, Carnevali, & Marchelli, 2009). The assay detected 10 cells in 25 g after
313
15 h enrichment. L. monocytogenes was also included in simultaneous detection of multiple food
314
pathogens from spiked milk and chicken meat samples by a chromogenic macroarray system, where biotin-
315
labelled PCR products were probed for further sensitivity (Chiang et al., 2012). A HtpG-like gene was used
316
as a target for L. monocytogenes. In 18 hours the system could detect 1 CFU/ml or g after an 8-h
317
enrichment step. This level was also achieved directly by PCR, but macroarray increased the sensitivity
318
when enrichment was not used. Naturally, the L. monocytogenes primers of the systems above could be
319
used separately for detecting only this species. The value of multiplexing might be limited by the different
320
enrichment (media) needs of target pathogens and by reduced sensitivity compared to using single primer
321
pairs (Wiedmann et al., 2014).
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2.2.5.2. Real-time PCR
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In real-time PCR the formation of amplification products can be monitored based on fluorescence as an
325
endpoint analysis or in real time. Another advantage over conventional PCR is the ability to quantify the
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ACCEPTED MANUSCRIPT start material. RT-PCR is also faster, and it is widely used in methods targeting food pathogens. An example
327
of endpoint analysis is identification of L. monocytogenes and five other Listeria species by RT-PCR (ssrA
328
gene) and high-resolution melting curve analysis (Jin et al., 2012). The sensitivity with L. monocytogenes
329
inoculated food samples was 102 CFU/ml, and the method was used for 30 artificially contaminated
330
samples (juice, milk, cheese, and meat) with a 93.3% correction rate.
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Real-time analysis is more common. A combined 24 h enrichment and RT-PCR method for L.
332
monocytogenes targeting the prfA gene gave DLs of 7.5 CFU/25 ml and 1–9 CFU/15 g in artificially
333
contaminated raw milk, and salmon, pâté, and cheese, respectively (Rossmanith, Krassnig, Wagner, & Hein,
334
2006). With 76 naturally contaminated food samples (fish; meat; meat and dairy products) 96% accuracy,
335
100% specificity and 76.9% sensitivity were achieved compared to the ISO standard. The method was also
336
applied for confirming the plating results during a multinational listeriosis outbreak (Schoder, Rossmanith,
337
Glaser, & Wagner, 2012). RT-PCR (actA gene) preceded by 24+6 h enrichment was evaluated against the
338
standard ISO method with 144 naturally and 61 artificially contaminated meat, fish, dairy, and vegetable
339
samples (Oravcová, Kuchta, & Kaclikova, 2007). Contamination of 1 CFU/25 g was detected, also from three
340
artificially contaminated samples that were negative with the ISO method. Enrichment (24+8 h) and RT-PCR
341
(here for ssrA) were also used for detecting L. monocytogenes in 175 samples taken from retail outlets and
342
food processing plants (O’Grady et al., 2009). The method had 99.44% specificity, 96.15% sensitivity and
343
99.03% accuracy compared to the ISO standard, and the DL was 1–5 CFU/25 g.
344
Rantsiou, Alessandria, Urso, Dolci and Cocolin (2008) developed and used L. monocytogenes qPCR (16S-23S
345
intergenic spacer) for 66 authentic food samples. Some positive results after 24 h enrichment were not
346
confirmed by cultural tests, which were interpreted as false negative. This was explained by the use of a
347
non-official isolation method and by competition from other species during the culture. Quantification limit
348
was 103–104 CFU/ml directly, but 10 CFU/ml could be detected after overnight enrichment. This RT-PCR was
349
also compared with the modified ISO method for the occurrence of L. monocytogenes in a dairy processing
350
plant (Alessandria, Rantsiou, Dolci, & Cocolin, 2010). Investigation of 200 samples (fresh cheeses, brine, and
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352
environmental samples where cells may be under stress conditions and possibly in VBNC state.
353
Use of a L. monocytogenes (hly gene) RT-PCR detection method developed earlier (Rodríquez-Lázaro et al.,
354
2004; Rodríquez-Lázaro, Pla, Scortti, Monzó, & Vázquez-Boland, 2005) has recently been evaluated or
355
validated in several food matrices. A validation trial of RT-PCR-based L. monocytogenes detection in
356
artificially contaminated soft cheese was reported by Gianfranceschi et al. (2014). Results from 11
357
European laboratories showed that RT-PCR with ISO compatible enrichment and DNA extraction performed
358
significantly better than the reference method (less false negatives and positives). It detected 10 CFU/25 g
359
in 27 h, and the relative accuracy was 82.75%, relative specificity 96.70%, and relative sensitivity 97.62%.
360
Also, the presence of L. innocua had a greater effect on the results obtained with the reference method
361
than by RT-PCR. In another study Rodríquez-Lázaro, Gonzalez-García, Gattuso, Gianfranceschi, and
362
Hernandez (2014) used this L. monocytogenes RT-PCR for artificially and naturally contaminated pork meat,
363
poultry meat, RTE lettuce salad, and sheep milk cheese samples. The best result was achieved when a 25 g
364
sample was diluted 1:10, enriched for 24 hours and DNA extracted using a commercial silica column. It
365
detected 2-4 CFU/25 g in 27 hours. Analytical performance with 200 natural samples was similar to that of
366
the reference method.
367
Some examples report that combining IMS and RT-PCR improves detection sensitivity. Use of nanoparticles
368
coated with anti-L. monocytogenes antibodies and RT-PCR (hlyA gene) was evaluated for detecting L.
369
monocytogenes in artificially contaminated milk (Yang, Qu, Wimbrow, Jiang, & Sun, 2007). Nanoparticles
370
performed better compared to Dynabeads, the sensitivity of the method being 226 CFU/0.5 ml. Duodu,
371
Mehmeti, Holst-Jensen, & Loncarevic (2009) evaluated filtration and IMS (Dynal anti-Listeria) in
372
combination with RT-PCR (hlyA) to detect L. monocytogenes in artificially contaminated hot-smoked
373
salmon. IMS reduced PCR inhibition significantly, and the DL was 20–40 CFU/g in a total of 3.5 h without
374
enrichment.
375
Several examples report differentiation of dead and live L. monocytogenes cells by RT-PCR. It was used for
376
differentiating viable (also VBNC) and dead L. monocytogenes cells in gouda-like cheese (Rudi, Naterstad,
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cells in spiked samples; 16–20 h enrichment and RT-PCR allowed detection of 10 CFU/g. Effect of
379
bacteriocin treatment on detecting L. monocytogenes by qPCR targeting hly was investigated using spiked
380
mixed salad samples (Cobo Molinos, Abriouel, Ben Omar, Martinez-Canamero, & Galvez, 2010). Above the
381
threshold level of 102 CFU/g the assay was better than the plating method for evaluating live cells but
382
below this level enrichment should be included. A filtering pre-treatment combined with quantitative RT-
383
PCR of the prfA gene enabled detecting 10 viable cells of L. monocytogenes in 10 g of spiked yogurt (D’Urso
384
et al., 2009) without enrichment. The method was in agreement with the ISO standard. Also Ye et al. (2012)
385
detected viable L. monocytogenes (1 CFU/ml) without enrichment in artificially contaminated frozen pork
386
by reverse transcriptase RT-PCR (hly). The method was recommended for showing the presence of live cells
387
but only for rough quantification of contamination.
388
RT-PCR has also been used in multiplex systems. An analysis consisting of enrichment (16–20 h), DNA
389
extraction and RT-PCR with dual-labelled probes or high resolution melting analysis was developed by
390
Omiccioli, Amagliani, Brandi, and Magnani (2009). It detected 1 CFU of each species – L. monocytogenes
391
(hlyA gene), E. coli, and Salmonella spp. – in 125 ml of spiked milk (divided into 25 ml aliquots). A multiplex
392
RT-PCR targeting L. monocytogenes (hly gene) and Salmonella spp. was described by Ruiz-Rueda, Soler,
393
Calvo, and Garcia-Gil (2011), and evaluated for L. monocytogenes with 54 naturally (dairy products;
394
vegetable, fish and meat matrices; RTE food) and artificially contaminated samples in <30 h. Sensitivity was
395
94.1% and efficiency 94.4% for L. monocytogenes compared to the ISO standard. The DL was 5 CFU/25 g
396
(for eggs and smoked salmon 102 CFU/25 g). Another assay for L. monocytogenes and Salmonella spp. was
397
developed by Garrido et al. (2013) and used for 63 spiked and 95 natural samples (fish, vegetables,
398
seafood, RTE, by-products, environmental). The detection limit was 5 CFU/25 g after 24±2 h and 8 h
399
enrichments, and the quality parameters >90%. A simultaneous enrichment (18 h) and detection of L.
400
monocytogenes (2 CFU/g) with Campylobacter, Salmonella, and E. coli in 24 h was developed and used for
401
212 routine samples (Köppel, Kuslyte, Tolido, Schmid, & Marti, 2013) in concordance with the ISO method.
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Terminology concerning the performance limits of the methods varies. Postollec et al. (2011) point out that
403
the DL means the minimum number of microbes that can be detected, but the quantification limit is the
404
lowest amount that can be accurately quantified and is usually higher than DL. Also, the limits obtained in a
405
food matrix should be used instead of limits in pure cultures.
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408
Most isothermal amplification-based methods for Listeria employ the LAMP technique, which uses several
409
primer pairs in a reaction performed at constant temperature, avoiding the need for special instruments.
410
Compared to PCR and RT-PCR other advantages include high specificity, speed and tolerance of sample
411
impurities (Niessen, Luo, Denschlag, & Vogel, 2013). Also, RNA can be used directly as a target in the
412
reaction for increased sensitivity.
413
A LAMP assay for L. monocytogenes was developed by Wang, Huo, Ren, and Li (2010) using primers based
414
on iap (P60 extracellular protein, invasion associated protein IAP) and evaluated with 125 naturally
415
contaminated milk samples. The method was 100% in concordance with the ISO standard when a 6-h
416
enrichment was included. The DL of the assay was 186 CFU/ml or 8-10 cells/reaction. Wan et al. (2012)
417
used propidium monoazide with LAMP based on the hlyA gene for detecting viable L. monocytogenes in
418
spiked chicken, pork, ground beef, and milk powder. The DL of the system was 102 CFU/ml in pure culture
419
and 3.1 x 103 CFU/g in all tested food types. A LAMP assay based on L. monocytogenes prfA together with
420
12/24 h enrichment was used for artificially contaminated milk (Cho, Dong, Seo, & Cho, 2014) and it
421
detected 2.22 x 101–2.22 CFU/ml L. monocytogenes. In pure culture the DL was 2.22 x 102 CFU/ml. A
422
commercial LAMP kit for Listeria based on sigB sequences (3M Molecular Detection Assay Listeria) was
423
validated against two cultural methods (3M Modified Listeria Recovery Broth and FDA BAM standard
424
method) with 391 environmental sponge samples from retail, and meat, dairy, and seafood processing
425
plants (Fortes, David, Koeritzer, & Wiedmann, 2013). The results after 22 h enrichment were not different
426
from the culture methods, and they could be obtained in 24 hours. Other, non-validated LAMP kits are also
427
available. Another type of isothermal amplification, hyperbranching rolling circle amplification (HRCA),
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based on hly gene and gold nanoparticle-based colorimetric assay was demonstrated for L. monocytogenes
429
detection in artificially contaminated milk (Fu, Zhou, & Xing, 2013). The sensitivity of the assay was 100 aM
430
target gene or 75 copies of genome DNA/reaction. In general, isothermal methods are not yet as sensitive
431
as PCR or RT-PCR, but they are being improved. More thorough validation testing is also needed.
433
2.3. Characteristics of various detection methods
434
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Conventional (culture and chromatographic) techniques are based on phenotypic tests. Their advantages
436
include sensitivity, reliability in efficiency, applicability to food samples, and low cost, but a major
437
disadvantage is the long time to result, in addition to labour intensiveness and lack of specificity (Jasson et
438
al, 2010; Yeni, Acar, Polat, Soyer, and Alpas, 2014).
439
The specificity of immunological methods relies on antigen-antibody binding and may not always be high
440
enough. They are also less sensitive than nucleic acid-based methods (Yeni et al., 2014). On the other hand,
441
these tests can be automated and are fast, reproducible, and less sensitive to food interference (Jasson et
442
al., 2010).
443
Compared to the other techniques, nucleic acid-based methods are more specific, using DNA or RNA
444
sequences as targets. RNA instead of DNA provides more sensitivity and information about the viability of
445
the bacteria (Jasson et al., 2010). On the other hand, because rRNA is rather stable and degrades slowly
446
after bacterial cell death, assays targeting rRNA may give false positives whereas mRNA-based tests are less
447
likely to do so (Wiedmann et al., 2014). Molecular tests are rapid and reproducible, enable multi-parameter
448
testing, and can be automated (Jasson et al., 2010; Yeni et al., 2014). The drawbacks include higher costs
449
and the negative effect of the food matrix on performance, especially with PCR (Jasson et al., 2010; Yeni et
450
al., 2014). Yet, Rodriguez-Lavaro et al. (2014) calculated that the RT-PCR method (EUR 3) is cost-effective
451
compared to the standard method (EUR 15). De Medici et al. (2014) also came to the same conclusion,
452
considering all analysis costs.
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ACCEPTED MANUSCRIPT In addition to the detection step, most methods include sample preparation or enrichment steps, which all
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have an impact on the final result. Likewise, the type of food, other microbes in food samples, and the
455
stress conditions on the target pathogen (e.g. sub-lethally injured cells) all affect the test performance
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(Jasson et al., 2010).
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The most rapid alternative methods have been performed with a food matrix within 1-3.5 hours with the
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DLs ranging from 10–40 CFU/g to 102-103 CFU/ml without any enrichment (Table 1). The sensitivity of the
459
methods is usually increased by enrichment. Using RT-PCR, a level of 1–5 CFU/25 g after 20–30 h culturing
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has been reported (Table 1). At this detection level the shortest enrichment times were 3–6 h (Zeng et al.,
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2006). Also methods without any enrichment steps have been reported.
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3. Validated and non-validated alternative detection methods
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The Food Safety and Inspection Service (FSIS) (FSIS, 2014) provides a list of foodborne pathogen test kits
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validated by independent organizations (AOAC, AFNOR, MicroVal or NordVal). Currently over 90 detection
467
methods are listed for Listeria spp. and L. monocytogenes. The same kit can be validated separately for
468
different matrices or purposes (detection, enumeration). One third of the tests target L. monocytogenes.
469
An Internet search was performed for commercially available, but non-validated alternative tests; the
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results are presented in Table 2. The non-comprehensive list of nearly 40 tests shows that, in addition to
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the officially approved tests, there exists a wide range of methods for the detection of Listeria, mostly
472
based on some amplification method. Half of them are L. monocytogenes specific.
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Detection kits based on amplification and hybridization techniques are the most numerous (43%) among
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the validated Listeria/L. monocytogenes alternative methods, followed by immunological (31%) and culture
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(26%) methods. Nucleic acid technology, especially RT-PCR, is the most commonly found type also in the
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case of non-validated commercial tests.
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The fastest total analysis times given are 16–24 h for the ANSR Listeria test (Neogen Corporation), 20.5 ± 2
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h for the ADIAFOOD Listeria and L. monocytogenes tests (bioMérieux SA) and 20 ± 2 h for the GeneDisc
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ACCEPTED MANUSCRIPT Listeria monocytogenes test with raw milk (Pall Corporation), which are all based on nucleic acid
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amplification. RapidChek Listeria (Romer Labs) and Vidas LMX Listeria monocytogenes (bioMérieux SA) are
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immunological tests claimed to yield results in 24 hours. A new Sample6 DETECT/L (Sample6) for Listeria
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environmental samples has recently received an AOAC certification. The test utilizes bacteriophages and is
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stated to yield results in < 8 hours without enrichment.
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4. Detection methods used by the food industry
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Although rapid or alternative methods are increasing in number and have been commercially available for
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many years, it has not been evaluated in the literature how widely they are actually being used in the food
489
industry.
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Some information is, however, given on the topic. According to a review by The Food Standards Agency in
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the UK (FSA, 2005) on the microbiological methods used by the food industry or laboratories, the most
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popular food categories tested were milk and dairy products and meat, poultry, and related products.
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Conventional standard culture methods were the most common category, out of which the aerobic
494
mesophilic plate count method (30 °C) was used by 84% of the laboratories. The most common externally
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performed test type was pathogen testing, and of the tests for specific bacteria carried out in-house, the
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most popular were tests for Staphylococcus aureus and other coagulase positive staphylococci (CPS),
497
Salmonella, Listeria spp., Bacillus cereus, and L. monocytogenes.
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Only a minority of companies had replaced the traditional methods with alternative ones. The reluctance to
499
adopt new techniques was attributed to the cost of capital equipment and consumables, insufficient
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validation data, the need to demonstrate the suitability of alternative methods in-house, and lack of
501
knowledge in connection with molecular methods. A majority of 71% of laboratories were not planning to
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adopt new methods. The report was based on surveys carried out in 1998–2001, so it is assumed that the
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alternative methods have gained wider usage since then.
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ACCEPTED MANUSCRIPT A more recent survey has been carried out by MoniQA: Monitoring and Quality Assurance in the Total Food
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Supply Chain, an EU project collecting information on the use of and future needs for analysis methods,
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including rapid methods, in 11 EU member countries and 6 non-EU members (Lebesi, Dimakou, Alldrick, &
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Oreopoulou, 2010; Lebesi, Bilbao, Diaz, Papadaki, & Oreopoulou, 2011). The samples examined most often
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were final products (94% of the respondents) and raw materials (90%), followed by environmental samples
509
(69%) and intermediate products (59%) (Lebesi et al., 2010). The largest respondent groups in terms of
510
product categories were meat and fish, beverages, dairy, and fruit/vegetable companies. Nine per cent
511
performed the analyses only in-house, 35% used only external laboratories, and 56% had tests performed
512
both internally and externally. Microbiological contaminants accounted for 90% of the analytes tested.
513
Of the respondent food companies performing analyses in-house, two-thirds used rapid methods in daily
514
operations, most commonly for microbiological analytes. The most widely used rapid tests were for E. coli
515
(~32%), total bacteria (~23%), and Salmonella (~18%). Rapid Listeria tests were used by about 12% of
516
respondents and 16% stated a need for one (Lebesi et al., 2011). The number of non-validated rapid
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microbiological methods was presented to be around 160, the number of validated methods more than 50,
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and the number of rapid methods used by the food industry more than 40.
519
Almost all respondents expressed an interest in extending the range of tests performed, and in terms of
520
their future needs, most ranked rapid microbiological tests as a priority. The introduction of rapid methods
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was seen as an improvement in food safety follow-up (62% of the respondents). In all, new rapid methods
522
were already in wide use in the food industry or the companies were ready to implement them.
523
Information on the use of different microbiological methods in the food industry can also be obtained from
524
market reports. Recent reports reveal similarities and differences in food safety testing geographically. In
525
15 years the total test volumes have increased by 128 per cent, with pathogen testing taking a bigger
526
portion of the total volume (from 13.7% to 23.2%) (Weschler, 2013). In the US 78% of the tests are for
527
routine and 22% for specific pathogens, while in the EU the figures are 82% and 18%, respectively
528
(Weschler, 2012). The organism types (Total Viable Organisms (TVO), Coliforms, Yeast/Mould,
529
Staphylococci), tested routinely are similar in both the USA and EU but the tested specific pathogens differ:
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ACCEPTED MANUSCRIPT in the USA, tests for Listeria spp. (41%) and E. coli O157 (11%) are more common, whereas in the EU L.
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monocytogenes is tested much more often (34% in EU vs. 3% in USA), with E. coli tests amounting to just
532
1.8%. Salmonella is tested equally in both regions.
533
Raw material samples account for 26% of all food samples collected (in the USA 8%, EU 16%), in-
534
process/environmental samples for 25% (USA 44%, EU 26%), and end products for 49% (USA 48%, EU 59%),
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so there is significant variation geographically (Weschler, 2013). This reflects the different trends and
536
perceptions about food safety.
537
The used detection methods also vary according to geographical location. In the USA, mostly convenience
538
methods are used for routine testing (66% in USA vs. 23% in EU), whereas in the EU traditional tests prevail
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(65% in EU vs. 20% in USA) (Weschler, 2012). In pathogen testing, the USA uses antibody- and molecular-
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based methods (together 88%), while the EU relies on traditional or convenience culturing (66%).
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5. Future trends
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In addition to attempts to improve or modify the existing food pathogen detection methods, new
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technologies are being developed. For example a proof-of-concept study reported by Chai, Horikawa, Li,
546
Wikle, and Chin (2013) introduced a phage-coated magnetoelastic biosensor for detecting Salmonella
547
bacteria on fresh food surfaces in real time and in-situ. The DL of the system was < 1.5 x 103 CFU/mm2.
548
Another trend seen in the literature is the simultaneous targeting of multiple pathogens during or in one
549
assay, be it immunological or based on some form of amplification or sensor method. L. monocytogenes is
550
frequently being identified together with other food pathogens, especially Salmonella and E. coli.
551
Methods based on isothermal amplification, e.g. LAMP, are increasing in the literature (Niessen et al.,
552
2013), also for L. monocytogenes, and detection kits relying on the same technology are emerging in the
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market (Table 2). According to Niessen et al. (2013) food safety and quality laboratories in the public sector
554
and industry are already using LAMP or planning to use it.
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ACCEPTED MANUSCRIPT Some hints or indications of future trends concerning microbiological methods might be seen in the
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patenting statistics. Therefore, we carried out a worldwide survey on the Espacenet database for the
557
detection, identification or determination of Listeria, which yielded about 120 references from the years
558
2000 to 2014. Out of these, 65% are from the Far East (44% from China alone) reflecting the current trend
559
in assigning countries. The USA is the next with 15%, followed by Korea (10%), Japan (8%) and France (6%).
560
Most applications/patents were based on PCR or other amplification technologies, followed by
561
immunoassays and other types of methods. Amplification-based methods were also the most numerous
562
category in patent publications from Europe and the USA, but the second place was held by inventions
563
concerning the culture of Listeria.
564
Nucleic acid-based Listeria detection methods account for a much bigger portion of patents than of
565
currently validated alternative test kits (70% vs. 43%). Likewise, the non-validated alternative methods are
566
mostly nucleic acid amplification or probing methods (Table 2). It is therefore anticipated that the list of
567
validated alternative methods will also be dominated by nucleic acid methods in the near future.
568
Based on their recent reports, Strategic Consulting, Inc. (SCI) (SCI, 2014) suggests some current trends in
569
food safety testing. First, food testing will increase because of public concern, active media, and increasing
570
regulations, but the use of rapid methods will grow at a different pace in different areas. Second, food
571
safety testing is shifting to third-party laboratories due to the growing amount of effort and investments
572
needed to carry out the analyses in-house, in addition to the expectations of accreditation. Third, more
573
environmental testing, especially for pathogens, will be carried out at food production plants because of
574
new regulations.
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6. Conclusions
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There exists a wide selection of methods for targeting L. monocytogenes and Listeria spp. that can be
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concluded in 48 hours. The number of validated methods is close to one hundred, and additionally tens of
580
other commercial methods are on the market. The alternative methods in the recent literature rely on
24
ACCEPTED MANUSCRIPT various amplification methods, with the number of isothermal applications increasing. The sensitivity levels
582
of the standard culture methods have also been achieved by different alternative technologies but in a
583
shorter detection time. Particularly RT-PCR-based detection of authentic food samples is rivalling the
584
standard culture methods in terms of the detection limit. In addition, the enrichment time is shortening or
585
enrichment is not included in new methods. Especially the different sensor-based approaches with an
586
analysis time of a few hours favour direct detection, but in general their DLs do not yet quite match those
587
of amplification methods. Some of the rapid tests in the literature have been thoroughly validated with
588
reference strains and different food matrices, but mainly they are more or less demonstrative and need
589
further evaluation. The users of the methods, laboratories in the industry and the public sector, are slowly
590
but steadily adopting rapid methods for use along with the standard ones or even replacing them.
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7. Acknowledgements
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This work was supported by the project A31588 (Food Safety Cluster) funded by the Council of Oulu Region
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from the European Regional Development Fund (ERDF) of the European Union.
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1 Table 1 Detection limits of the standard and some alternative, rapid Listeria monocytogenes (LM) identification methods Detection limit
Matrix
Enrichment | Total time if given
ISO 11290 FDA USDA-FSIS
<1 CFU/25 g <1 CFU/ml <1 CFU/g in 25 g
all foods dairy, fruit & vegetable, seafood meat, poultry, egg, environmental
96/120 h|4–7 d 76/100 h|4–7 d 66–74/90–98 h|4– d
RNA RT-PCR IMS+RT-PCR Multiplex-RT-PCR
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ISO, 2004a,b Hitchins & Jinneman, 2013 FSIS, 2013
6 h|2 d
5/2/9
Kretzer et al., 2007
14 h|15 h
11/5/13
Shim et al., 2008
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lettuce, cheese, fish, meat, milk (A/N 275 ) meat (A 15/N 116)
10 CFU/ml 20 CFU/ml 5–10 CFU/g <5 CFU/ml 20–50 CFU/ml 2 >10 CFU/g
2
milk (A) cheese, fish, milk, strawberries, swab (N 22) meat, seafood, dairy products (A 30/N 190) milk (A) meat, lettuce (A) vegetables (N 191)
No|2 h 11 h|16 h 24 h| 2 h at RT|4 h No|2 h 7 h|
1/-/3 1/-/2/4/38 1/-/2 1/-/2 2/-/-
Cho & Irudayaraj, 2013 Jaakohuhta et al., 2007 Portanti et al., 2011 Cho I. et al.,2014 Wang et al., 2011 Moreno et al., 2012
1 CFU/g 8 CFU/ml c 1.45 CFU/ml 10 CFU/25 g 1 CFU/ml or g 1 CFU/15 g 2–4 CFU/25 g 1 CFU/25 g 1–5 CFU/25 g
sausage, cheese (A) cheese, meat(A) milk, sewage water, vessel surfaces (A/N 27) liquid egg (A) milk, meat (A) cheese, pâté (A/N 76) meat, dairy products, vegetables (A) meat, fish, cheese, dairy products (A 61/N 144) meat, fish, dairy products, desserts (A 16/N 175) yogurt (A) meat (A) fish (A) milk (A)
24 h|2 d 5+17 h| 6 h| 15 h| 8 h|18 h 24 h| 24 h|27 h 24+6 h|2 d 24+4 h|2 d
1/-/1/-/5/6/2 1/-/2 15/8/262 100/30/29 d 9/-/d 1/-/d ?/-/-
Amagliani et al., 2006 Rip & Gouws, 2009 Zeng et al., 2006 Germini et al., 2009 Chiang et al., 2012 Rossmanith et al., 2006 Rodriquez-Lazaro et al., 2014 Oravcová et al., 2007 O’Grady et al., 2009
No| No| No|3.5 h 18±2 h|2 d
1/-/1/1/40 d 2/-/33/7/50
D’Urso et al., 2009 Ye et al., 2012 Duodu et al., 2009 Omiccioli et al., 2009
10 CFU/10 g 1 CFU/ml 10–40 CFU/g 1 CFU/5x25 ml
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10 CFU/10 g
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10 CFU/g
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Specificity tested LM/ Listeria spp./ other strains
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2 24 h|< 30 d 18 h|24 h No|2.5 h 24 h| No|
11/11/58 2/4/67 45/5/10 23/8/8 2/-/12
Ruiz-Rueda et al., 2011 Köppel et al., 2013 Wan et al., 2012 Cho A.R. et al., 2014 Wang, et al., 2010
blueberries (A) tomato extract (A) milk (A) meat (A)
No| No| No|1 h 18 h|24 h
3/-/4 1/-/1 ?/-/1/5/5
Davis et al., 2013 Radhakrishnan et al., 2013 Sharma & Mutharasan, 2013b Ohk et al., 2010
b
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Biosensors 2 Amperometric 10 CFU/g Impedance spectroscopy 4 CFU/ml 2 3 Piezoelectric 10 –10 CFU/ml 2 Fibre-optic 10 CFU/25 g a 0.1 CFU/g after 24 h enrichment
dairy products, fish, vegetables, meat (A/N 54) cheese, yogurt (A 250/N 212) meat, milk powder (A 4) milk (A) milk (A/N 125)
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5 CFU/25 g 2 CFU/g 3 10 CFU/ml e 2 CFU/ml 186 CFU/ml
A = tested artificially contaminated samples; N = tested naturally contaminated samples
c
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ACCEPTED MANUSCRIPT Table 2 Some commercially available non-validated L. monocytogenes and Listeria spp. tests Test type
Technical Service Consultants Ltd Creative Diagnostics AdipoGen
Color
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HybriScan Listeria monocytogenes Listeria spp. tests Compact Dry LS Hygiena InSite™ Rapid Environmental Listeria Test Listeria Isolation Transwab
Path-Chek Hygiene Pathogen System Listeria Latex Agglutination Test Listeria Rapid Test Kit RIDASCREEN Listeria Singlepath® Listeria Listeria Detection Listeria Listeria spp. and Listeria monocytogenes Multiplex PCR with internal Control innuDETECT Listeria spp. Assay rapidSTRIPE Listeria Assay
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Immunochromatographic ELISA Immunochromatographic RT-PCR RT-PCR RT-PCR RT-PCR RT-PCR
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Liferiver/BioSB NORGEN Biotek Corp. Diatheva Diatheva Q-Bioanalytic GmbH
RT-PCR PCR/RT-PCR PCR RT-PCR PCR/RT-PCR
R-Biopharm AG
RT-PCR
SA Scientific Eiken Chemical CO. Ltd. / Mast Group Ltd. 3M
LAMP, Real time LAMP, Real time
TwistDx
Sigma Aldrich
IAMP (Recombinase) Realtime Sandwich hybridization
R-Biopharm AG Hygiena
Color Color
Medical Wire & Equipment Microgen Bioproducts Creative Diagnostics Hardy Diagnostics R-Biopharm AG Merck IDLabs BioGX GEN-IAL GmbH
Color
Analytik Jena Analytik Jena
RT-PCR PCR & lateral flow
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Listeria Monocytogenes Rapid Test Listeria monocytogenes SURE mono ELISA Kit Singlepath® L’mono BactoReal Listeria monocytogenes genesig Listeria monocytogenes Listerfast Listeria monocytogenes Listeria monocytogenes with internal Control Listeria monocytogenes Real Time PCR Kit Listeria Monocytogenes PCR Detection Kit Listeria monocytogenes PCR detection Kit Listeria monocytogenes FLUO kit QuickBlue (RealQuick) Listeria monocytogenes SureFood® PATHOGEN Listeria monocytogenes PLUS Listeria monocytogenes Detection Kit Loopamp Listeria monocytogens DetectionKit Molecular Detection Assay Listeria monocytogenes TwistAmp® exo+ListeriaM
Manufacturer/Supplier
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Test Listeria monocytogenes tests SwabSURE Listeria P (also L. ivanovii)
LAMP, Real time
Color Latex Agglutination Immunoassay Immunoassay Immunochromatographic PCR RT-PCR RT-PCR
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PCR/RT-PCR RT-PCR Probe & fluorescence microscopy
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QuickBlue (RealQuick) Listeria spp. SureFood® BAC Listeria Screening PLUS VIT® Listeria
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Highlights: • overview of currently available, rapid (<48 h) L. monocytogenes detection methods • focus on naturally or artificially contaminated food and environmental samples • summary of the most rapid and sensitive methods • many methods as sensitive as standard methods, but much faster
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