Detection of Listeria monocytogenes and the toxin listeriolysin O in food

Journal of Microbiological Methods 64 (2006) 141 – 170 www.elsevier.com/locate/jmicmeth

Review

Detection of Listeria monocytogenes and the toxin listeriolysin O in food Robin L.T. Churchill, Hung Lee *, J. Christopher Hall Department of Environmental Biology, University of Guelph, Guelph, ON, Canada N1G 2W1 Received 29 March 2005; received in revised form 14 October 2005; accepted 14 October 2005 Available online 28 November 2005

Abstract Listeria monocytogenes is an emerging bacterial foodborne pathogen responsible for listeriosis, an illness characterized by meningitis, encephalitis, and septicaemia. Less commonly, infection can result in cutaneous lesions and flu-like symptoms. In pregnant women, the pathogen can cause bacteraemia, and stillbirth or premature birth of the fetus. The mortality rate for those contracting listeriosis is approximately 20%. Currently, the United States has a zero tolerance policy regarding the presence of L. monocytogenes in food, while Canada allows only 100 cfu/g of food. As such, it is essential to be able to detect the pathogen in low numbers in food samples. One of the best ways to detect and confrim the pathogen is through the detection of one of the virulence factors, listeriolysin O (LLO) produced by the microorganism. The LLO-encoding gene (hlyA) is present only in virulent strains of the species and is required for virulence. LLO is a secreted protein toxin that can be detected easily with the use of blood agar or haemolysis assays and it is well characterized and understood. This paper focuses on some of the common methods used to detect the pathogen and the LLO toxin in food products and comments on some of the potential uses and drawbacks for the food industry. D 2005 Elsevier B.V. All rights reserved. Keywords: Detection methods; ELISA; Enrichment; Foodborne pathogen; Listeria monocytogenes; Listeriolysin O; PCR

Contents 1. 2.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods to detect L. monocytogenes and LLO . . . . . . . . . . . . . . . . . . . . . 2.1. Food processing prior to detection . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Immuno-separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Culture-dependent enrichment methods for detection of L. monocytogenes . . . . 2.3. Automated methods for detection of L. monocytogenes (non-nucleic acid-based) 2.4. Nucleic acid-based methods for detection of L. monocytogenes . . . . . . . . . 2.4.1. PCR-based detection of L. monocytogenes . . . . . . . . . . . . . . . . 2.4.2. NASBA — nucleic acid sequence-based amplification. . . . . . . . . . 2.4.3. DNA microarrays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Pathogen subtyping and verification methods . . . . . . . . . . . . . . . . . . . 2.5.1. RFLP — restriction fragment length polymorphism . . . . . . . . . . .

* Corresponding author. Tel.: +1 519 824 4120x53828; fax: +1 519 837 0442. E-mail address: [email protected] (H. Lee). 0167-7012/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2005.10.007

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2.5.2. Ribotyping . . . . . . . . . . . . . . . . . . . . . . . 2.5.3. PFGE — pulsed-field gel electrophoresis . . . . . . . . 2.5.4. AFLP — amplified fragment length polymorphism . . . 2.5.5. RAPD — randomly amplified polymorphic DNA . . . 2.5.6. FISH — fluorescence in situ hybridization . . . . . . . 2.6. Immunoassay-based methods for detection of L. monocytogenes 2.6.1. Types of antibodies used in detection of pathogens . . . 2.6.2. ELISA — enzyme linked immunosorbent assay . . . . 2.6.3. ELFA — enzyme-linked fluorescent assay . . . . . . . 2.6.4. Immunoprecipitation and agglutination assays . . . . . 3. Future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Listeria monocytogenes is a foodborne pathogen that causes listeriosis. In the environment, the pathogen is most commonly isolated from soil and silage. In foods, it has been found in raw or processed food samples including dairy products, meat, vegetables and seafood (Gugnani, 1999; Meng and Doyle, 1997). Between 1979 and 1999 in the United States and Canada, there were 6 major outbreaks of listeriosis. These outbreaks were associated with eating such diverse foods as lettuce, carrots, commercially prepared coleslaw, pasteurized 2% milk (contaminated after pasteurization), paˆte´, pork tongue in jelly, and soft cheeses made from raw milk, chocolate milk and hotdogs (Donnelly, 2001). In infected individuals, listeriosis is characterized by meningitis, encephalitis, and/or septicaemia. Less commonly, infection will result in cutaneous lesions and flu-like symptoms (Meng and Doyle, 1997). In pregnant women, the pathogen can cause bacteraemia which, if left untreated, can lead to amnionitis and infection of the fetus, resulting in stillbirth or premature birth. The mortality rate for those contracting listeriosis is approximately 20% (Gugnani, 1999; VazquezBoland et al., 2001). Infection by L. monocytogenes is mediated by a number of virulence factors. The most important of these is listeriolysin O (LLO), a pore-forming toxin secreted by the pathogen. LLO is absolutely required for virulence by L. monocytogenes, and is found only in virulent strains of the species. In addition, being a secreted protein, its detection can serve as an indicator of the presence of L. monocytogenes in a food sample. There are a number of papers covering the biology of L. monocytogenes and the physiology of the LLO toxin. For L. monocytogenes, please see the review papers by Vazquez-Boland et al. (2001), Donnelly

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(2001), and Kathariou (2002). For LLO, reviews by Alouf (1999) and Jacobs et al. (1999) are particularly informative. This paper will focus on the detection of L. monocytogenes and its toxin, LLO in contaminated food samples. 2. Methods to detect L. monocytogenes and LLO Although L. monocytogenes is found ubiquitously in the environment, it is a concern mostly because of its role as a foodborne pathogen. Presently in the United States, there is a zero tolerance policy for the levels of L. monocytogenes allowed in food (McLauchlin et al., 2004). In Canada, the limit is 100 cfu/g in ready-to-eat foods (Health Canada, 2004). This means that food producers must be able to detect low levels of the pathogen quickly and accurately to avoid the large costs and legal repercussions involved in a recall of food due to L. monocytogenes contamination. The ideal pathogen detection test for the food industry should have a number of attributes. The optimal method for pathogen detection should be simple to perform. It should be sensitive enough to detect pathogens at levels as low as 1 cell/g of food material. L. monocytogenes has an unknown infectious dose that could be as low as 102 microorganisms in some cases (McLauchlin et al., 2004). The method should be specific to the pathogenic species within the genus; in Listeria for example, there are six species, of which only L. monocytogenes is an important human pathogen. The method should be rapid, giving results in less than a day. Finally, the method should be amenable to automation and inexpensive (Ingianni et al., 2001). In the following sections we describe some of the commonly used techniques to detect L. monocytogenes. It should be noted that none of the methods alone satisfies all of the criteria listed above.

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2.1. Food processing prior to detection All the L. monocytogenes detection methods available today require a large number of pathogenic organisms to identify the presence or to quantify the number of organisms present in the sample. This holds true whether the detection method is culture-, nucleic acid-, or immunoassay-based. To detect pathogens in low numbers, researchers must first process the food to make the pathogen available for detection and must also employ an enrichment strategy to selectively increase the number of pathogens prior to using whatever detection method they have chosen. The enrichment steps generally take between 24 and 72 h to complete and add significant amounts of time to the detection method, making this step a prime candidate for alteration in attempts to lower the detection time of pathogens in food. In general, processing begins with taking a sample of the food or beverage. For solid foods, the sample is added to a sterile stomacher bag along with some enrichment media. The contents of the bag are homogenized so that all of the food molecules are in association with the media, allowing any bacteria to be washed into the media. The homogenate is incubated for up to 24 h to allow growth of the bacteria. After incubation, an aliquot of the homogenate is taken for use in further enrichment steps, selective culturing of specific organisms, or isolation of components for the pathogen detection test (Hayes et al., 1992; Ryser et al., 1996). Liquid cultures are treated in a similar manner, except that the sample is added directly to the enrichment media without the need for prior homogenization (Kells and Gilmour, 2004). 2.1.1. Immuno-separation Immuno-separation makes use of antibody specificity towards a pathogen to concentrate that pathogen before other methods are used to amplify and identify the bacteria. Antibodies are attached to beads, and added to a homogenized sample. Any pathogen with affinity for the antibody should attach to the bead complex. The beads are then separated from the slurry through either the use of a magnet (immunomagnetic separation with magnetic beads (Hudson et al., 2001)), or through centrifugation (protein-A-linked sepharose beads (Gray and Bhunia, 2005)). In theory, the technique should concentrate pathogens, thus making detection a feasible option without the requirement for the long enrichment incubations required to amplify pathogen numbers to a detectable level.

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Hudson et al. (2001) used immunomagnetic separation to isolate L. monocytogenes directly from ham. In this procedure, the food was homogenized with some growth media, the particulate matter removed, and after a number of washes, particles of bacterial size were pelletted and resuspended in a small volume of buffer. Commercial immunomagnetic beads coated with an anti-Listeria sp. antibody were added to the buffer solution and incubated to allow binding of the L. monocytogenes to the beads. The beads were trapped on a magnet, washed, and the DNA extracted for amplification of L. monocytogenes-specific genes by PCR. The immunomagnetic separation and concentration procedures reduce the detection time to about 1 day, at least for ham, but was limited in terms of sensitivity, since the recovery of cells on the beads was only about 20% of those initially added (Hudson et al., 2001). Immunomagnetic separation on average allowed detection of 1–2 cfu/g food sample, but the results were somewhat variable in terms of sensitivity, having a detection limit from 0.1 cfu/g to greater than 5.7 cfu/g (Hudson et al., 2001), making this method promising, but not ready to be used by the food industry until the efficiency of immunomagnetic isolation is improved. 2.2. Culture-dependent enrichment methods for detection of L. monocytogenes Most conventional methods for detecting foodborne bacterial pathogens in food and other substrates rely on the use of microbiological media to selectively grow and enumerate bacteria. The methods are sensitive, inexpensive and provide qualitative as well as quantitative results. Unfortunately for the food industry, where time and costs are issues, preparation of media and plates, as well as colony counting and biochemical characterization of the isolated colonies, make for a time-consuming and labour-intensive process (de Boer and Beumer, 1999). The success of culturing methods depends on the number and state of the bacteria in the sample, the selectivity of the media (balance between inhibition of competitors and inhibition of the target organism), the conditions of the incubation (time, temperature, O2), and selectivity of the isolation medium (distinction between the target organism and competitive microflora) (Beumer and Hazeleger, 2003). The most common methods used to detect L. monocytogenes are those developed by the US Department of Agriculture–Food Safety and Inspection Service (USDA–FSIS) for the detection of L. monocytogenes in meat and poultry products (Silbernagel et al., 2004; Wallace et al., 2003), the Federal Drug Administration

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Table 1 Comparison of common culture-based methods for L. monocytogenes detection in foods Method name

Recommended foods for method

Steps to purification

Novel aspects of method

Detection limit

FDA

–Dairy –Fruit and vegetable –Seafood

Identification after enrichment requires colony morphology, motility, haemolysis, catalase activity, nitrate reduction and other testing for confirmation of species (Hitchens, 1995)

b0.7 cfu/ml goat’s milk (Lammerding and Doyle, 1989)

ISO-11290-1

–All foods

The use of ALOA plates allows L. monocytogenes to be distinguished from other Listeria spp. (Beumer and Hazeleger, 2003)

5–100 cfu/25 g. However, the presence of L. innocua led to a number of false negative results (Scotter et al., 2001)

NGFIS

–All foods

1) Growth in FDA enrichment broth (FDA–EB) containing 40 mg/L naladixic acid, 15 mg/L acriflavine, 50 mg/L cycloheximide HCl for 24 h 30 8C 2) FDA–EB 48 h 30 8C 3) Plating on Oxford and LPM (see USDA–FSIS method) or PALCAM (see NGFIS method) agars for easier distinction of Listeria colonies (Jinneman et al., 2003) 4) Biochemical and metabolic assays to confirm identity 1) Growth in half Fraser broth for 24 h at 30 8C 2) Growth in Fraser broth containing 0.5 g/L ammonium iron (III) citrate, 12.5 mg/L acriflavine, 10 mg/L nalidixic acid for 48 h at 37 8C 3) Isolation on PALCAM (see NGFIS method) or Agar Listeria Ottavani and Agosti (ALOA) plates containing 20 mg/L nalidixic acid, 0.02 mg/L ceftazidime, 76700 U/L polymyxin B, 50 mg/L cycloheximide, 10 mg/L amphotericin B (Hitchens, 1995) 1) Growth in a nutrient broth such as Listeria enrichment broth (LEB) #1/#2 for up to 48 h at 30 8C 2) Subculture onto PALCAM agar containing 10 mg/L polymyxin B, 5 mg/L acriflavine, 15 g/L LiCl, 20 mg/L ceftazidimine, 0.8 g/L esculin, 0.5 g/L ferric ammonium citrate, 10 g D-mannitol, 80 mg/L phenol red for 48 h at 30 8C

–PALCAM agar contains esculin and a ferrous salt to make colonies appear grey with a sunken black centre; D-mannitol and phenol red can differentiate between esculin positive Enterococci spp. and L. monocytogenes (van Netten et al., 1989)

b10 cfu/g in minced meat, fermented sausage, broiler carcasses, mush rooms, soft cheese (van Netten et al., 1989)

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Table 1 (continued) Method name

Recommended foods for method

Steps to purification

Novel aspects of method

Detection limit

USDA-FSIS

–Meat –Poultry

–As of 2002, the USDA–FSIS accepts the use of the BAXR system screen for L. monocytogenes rather than using biochemical assays (http://www. fsis.usda.gov/OA/ news/2002 /bax.htm)

b0.7 cfu/ml goat’s milk (Lammerding and Doyle, 1989)

Cold Enrichment

–All foods

1) LEB #1 containing 20 mg/L nalidixic acid, 12 mg/L acriflavine for 24 h at 30 8C 2) LEB#2 + 20 mg/L nalidixic acid, 25 mg/L acriflavine HCl, 24 h, 308C 3) Plating on lithium chloridephenylethanolmoxalactam (LPM) agar for 48 h at 30 8C (Lammerding and Doyle, 1989), or BAXR testing (see text) 1) Grow in nutrient broth for 8 weeks at 4 8C 2) Plating on LPM agar for 48 h at 30 8C

–No antibiotics are used for selective enrichment –Temperature is the selective agent (Lammerding and Doyle, 1989)

b0.7 cfu/ml goat’s milk (Lammerding and Doyle, 1989)

(FDA) protocol for detecting the organism in dairy (Kells and Gilmour, 2004; Lammerding and Doyle, 1989), fruit, vegetable and seafood products (Lammerding and Doyle, 1989; Norton et al., 2001; Thimothe et al., 2004), and the Netherlands Government Food Inspection Service (NGFIS), used for all foods (Donnelly, 2001; van Netten et al., 1989). These methods vary only in the type of selective media that are used (Hayes et al., 1992) (see Table 1). They are species-specific, but the strong selective media used may reduce growth of L. monocytogenes causing the methods to lose some of their sensitivity. For example, Hayes et al. (1992) showed that the USDA–FSIS method was only able to detect L. monocytogenes in 65% of foods known to be contaminated with this microorganism, while the NGFIS method only gave positive results for 74% of the same cases. If any two of the above three methods were used together, the percent success in detecting L. monocytogenes contamination increased to between 87% and 91%. This may still not be an acceptable level of detection, when deaths may result if the pathogen is present but not detected. Kells and Gilmour (2004) improved upon the detection rate of L. monocytogenes in milk using the FDA method by changing the final Oxford and PALCAM plating agar to L. monocytogenes Blood Agar (LMBA). LMBA has lower concentrations of some of the selective agents, so that haemolysis of the blood in the agar can be used to differentiate L. monocytogenes from other non-path-

ogenic species of Listeria. The change increased detection from 76.5% and 79.4% on Oxford and PALCAM agars, respectively, to 94.1%. As recently as 2002, the primary method for the detection of L. monocytogenes in food samples was based on traditional microbiological methods (Allerberger, 2003). Testing consists of three basic steps: enrichment, isolation, and confirmation (Buchanan, 1990). After enrichment in selective media, the culture is tested to determine if the physiology is typical of Listeria sp. Cells should be Gram-positive and rod shaped, show tumbling motility and positive catalase activity. The colonies should also display blue-grey appearance on Modified McBride Agar under oblique light (Benedict, 1990). If these criteria are satisfied, the culture is then tested for negative mannitol degradation and the ability to induce haemolysis on blood agar. Positive cultures must be further tested for negative xylose and positive rhamnose utilization before concluding that the sample is L. monocytogenes rather than one of the other Listeria species (Buchanan, 1990; Lovett, 1990). Other tests include positive esculin and tellurite usage, oxidase-negative status, and ability to produce acid from glucose. Even after all these tests, the serotype must still be determined (Buchanan, 1990). The type of media used for enrichment will affect the detection rates for L. monocytogenes. Brackett et al. (1990) investigated seventeen different media used for

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isolation and identification of L. monocytogenes for their efficacy of recovery from food and suitability for direct plating procedures. Of the seventeen media, nine were impractical for various reasons; for example, the media inhibited growth of L. monocytogenes to such an extent as to be useless, or it was difficult to distinguish L. monocytogenes colonies from the agar on the plates due to colour or size of the colonies. Of the eight remaining media, there were large differences in their ability to allow recognition or counting of L. monocytogenes, especially in the presence of competing microflora (Brackett et al., 1990; Golden et al., 1990). The type of food sample also had an effect on which type of enrichment media will yield the best results. For example, Rijpens and Herman (2004) found that non-selective enrichment was more effective for detecting L. monocytogenes in semi-hard or soft cheeses of the blue veined or red smear type, whereas for the mould-ripened soft cheeses, a selective enrichment medium was required. The need to optimize the detection media for each food type adds time to detection, and calls into question the effectiveness of the USDA–FSIS, FDA and NGFIS methods that are used for many different types of foods. There are a few problems with enrichment-based methods to detect L. monocytogenes in food samples. First, they are time-consuming. For example, the International Organization for Standardization (ISO) method 11290-1 (Wan et al., 2003), the official method used by Australian food inspectors, requires five days for determination of a negative result for L. monocytogenes contamination. If a positive test result occurs, additional days are required for biochemical tests to identify the species (Wan et al., 2003). Second, enrichment methods do not account for the recovery of sublethally injured bacteria that may be present as a result of heating, freezing or acidification of the foods (Brackett et al., 1990; Buchanan, 1990). These bacteria may not be able to form colonies under selective pressure during recovery, but are still virulent and able to grow in food. For example, sublethally injured L. monocytogenes cells are capable of repair, growth and subsequent virulence at storage temperatures of 4 8C or higher (Brackett et al., 1990; Donnelly, 2001) although the extent to which this occurs has been questioned (Foong and Dickson, 2004). Third, no single medium seems to be suitable for isolation of L. monocytogenes from a variety of food samples. The type of food, population of L. monocytogenes and other contaminants, and state of health of the L. monocytogenes isolates all affect which selection media will be the most effective (Brackett et al., 1990). Finally, there is evidence that some L. monocytogenes

strains are more sensitive to enrichment and selection procedures than others (Kathariou, 2002; Loncarevic et al., 1996; Ryser et al., 1996). For example, serotype 4b is prevalent in 50–70% of listeriosis outbreaks but is rarely found in contaminated food, thereby feeding speculation that its occurrence is under-estimated using this method. Related to this problem, the use of selective agents in media can favour the growth of other competitive species of Listeria. As an example, the common use of acriflavine in the enrichment media (to inhibit RNA synthesis and mitochondriogenesis) tends to affect L. monocytogenes more than L. innocua (Beumer and Hazeleger, 2003), leading to an underestimation of L. monocytogenes contamination. Acriflavine is used in both the USDA–FSIS and FDA methods of isolation. To avoid some of these problems, many newer methods rely on the initial use of selective, chromogenic substrates that change colours in the presence of some enzymes present only in certain species, making these species easier to identify. For instance, L. monocytogenes and L. ivanovii contain the plcA gene, encoding a phosphatidylinositol-specific phospholipase C, which is not found in any other Listeria species. When plated on selective BCM (Biosynth Chromogenic Medium) plates, positive colonies are turquoise, while all others are white (Jinneman et al., 2003). The two strains can then be separated based on sugar utilization or other plating techniques, thus reducing the number of tests that must be performed to detect L. monocytogenes in food samples (Jinneman et al., 2003). Some enrichment methods have also been modified or automated to increase throughput. For example, automated dilution of the samples before homogenisation and automated homogenisation in sterile bags is faster than sample preparation by hand (de Boer and Beumer, 1999). Flow cytometry has also been used to speed up counting times, but it cannot distinguish between living and dead cells (de Boer and Beumer, 1999). Another recent advance in culture methodology is the introduction of microplate techniques to monitor growth. Microplates are made up of 96 wells, and allow for many small (200 Al) cultures to be grown simultaneously. Growth can be measured over time by using a spectroscopic plate reader, obviating the need for sampling, and reducing the chance for contamination of the culture. Hora´kova´ et al. (2004) used this method to determine which of the selective media is best for growth of L. monocytogenes and which is the best for encouraging expression of virulence factors. They found that of six common media used for the

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enrichment of L. monocytogenes, brain heart infusion (BHI), Listeria enrichment broth (LEB) and glucose tryptone yeast extract broth encouraged the fastest growth of L. monocytogenes. 2.3. Automated methods for detection of L. monocytogenes (non-nucleic acid-based) A number of commercially available, semi-automated systems are available for the detection of various foodborne pathogens, including the MicroLog System (Biolog Inc., Hayward CA), the Microbial Identification System (MIS) (MIDI Inc., Newark DE), VITEK System (bioMerieux Vitek, Hazelwood, MO), and the Replianalyzer system (Oxoid Inc., Nepean, ON) (Odumeru et al., 1999). The Microlog System is based on carbon substrate oxidation profiles, MIS on lipid analysis and the VITEK and Replianalyzer Systems on biochemical selection. According to Odumeru et al. (1999), these systems are able to detect Listeria sp. reliably at the genus level 90–100% of the time, depending on the system being used, but biochemical tests are still required for subsequent detection at the species level. 2.4. Nucleic acid-based methods for detection of L. monocytogenes One of the biggest problems associated with detection of L. monocytogenes is the low numbers at which the bacteria are normally found in contaminated food samples (Hoffman and Weidmann, 2001). In one outbreak caused by consuming hot dogs in the United States, illness was attributed to contamination levels b 0.3 cfu/g of hot dog meat (Donnelly, 2001). DNA-based methods of detection employ ways of amplifying the specific genetic signals from a few cells. The following section summarizes some of the common ways in which nucleic acids have been used as detection targets for L. monocytogenes. A comparison of the various techniques is presented in Table 2. 2.4.1. PCR-based detection of L. monocytogenes Polymerase chain reaction (PCR) is the basis of many nucleic acid-based detection systems. With this method, total DNA is extracted from the food sample. Next, two oligonucleotide primers are selected that bind to a pathogen-specific target gene at opposite ends of opposing strands of DNA. A DNA polymerase is added along with the four types of deoxynucleotides (dNTPs) and appropriate buffers, and the mixture is inserted into

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a thermocycler. This machine cycles though a temperature regime that usually involves 94 8C to denature the DNA template, an annealing temperature of 50–65 8C to allow the oligonucleotide primers to bind to the gene, and 72 8C to allow extension by the DNA polymerase. The thermocycler runs through these temperatures a number of times to amplify the gene fragment exponentially. The resulting fragment is analysed by agarose gel electrophoresis after staining with ethidium bromide and visualisation under UV light. The presence of L. monocytogenes, both live and dead, can be detected by simply determining if a band representing the specific pathogen gene of interest is present. Performing PCR takes only a few hours, shortening the time required for pathogen detection dramatically, and the technique is conducive to automation and high throughput processing using 96-well plates. Aznar and Alarco´n (2002) and Shearer et al. (2001) claim that PCR-based detection of L. monocytogenes is more sensitive than culture-based methods for detecting the pathogen in contaminated food samples because more samples turned up positive in their tests for L. monocytogenes using the PCR method. The authors attribute the increase in sensitivity of the PCR method over culturing methods to the fact that the former does not have an initial selection step. The PCR method circumvents the problem that some cells do not grow in the selective media. The authors also claim that some of the false–negatives reported for culturing methods are due to methods that use colour changes to differentiate between species, and these methods are influenced by the subjectivity of the observer (Aznar and Alarco´n, 2002; Shearer et al., 2001). Among the target genes for PCR detection of L. monocytogenes are the hlyA gene (encoding LLO) (Blais et al., 1997; Hough et al., 2002; Hudson et al., 2001; Lehner et al., 1999; Lu¨beck and Hoorfar, 2003; Lunge et al., 2002; Norton et al., 2001; RodriguezLa´zaro et al., 2004a,b; Ryser et al., 1996; Thimothe et al., 2004; Weidmann et al., 1997), the iap gene (encoding an invasion-associated protein) (Cocolin et al., 2002; Schmid et al., 2003), inlB (encoding internalin B) (Ingianni et al., 2001; Jung et al., 2003; Lunge et al., 2002; Pangallo et al., 2001) and 16S rRNA (Call et al., 2003; Schmid et al., 2003). Between these genes, the most commonly used has been hlyA (Aznar and Alarco´n, 2002). Wan et al. (2003) compared the use of PCR to detect L. monocytogenes in salmon with the ISO culturing method 11290-1, and found the two methods gave comparable results in spiked samples if culture enrichment is used prior to PCR to lower the detection limit

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Table 2 Comparison of nucleic acid-based detection methods for L. monocytogenes Method of detection

Gene detected

Enrichment Total culture time detection time

Number of cells detected

Medium in which pathogens were detected

Ref.

PCR

Commercial kita inlAB inlAB hlyA

48 h None 16 h 96 h

58–60 h Not available 24 h ~100 h

2–8 cfu/25 g 105 cfu/ml 10 cfu/25 g N/A

Salmon Pure culture Frankfurter Smoked salmon

PrfA Commercial kit

40 h 72 h

~45 h 80 h

20–200 cells/25 ml Milk 0.5–3 cfu/25 g Frankfurter, soft cheese, smoked salmon, ground beef, radishes, frozen peas 103 cfu/0.5 ml Milk 1 cfu/0.5 ml Milk 2–10 cells/g Milk and various meat products 500 cfu/ml Pure culture 103–104 cfu/ml Skim milk 15 cfu Pure culture

(Wan et al., 2003) (Jung et al., 2003) (Jung et al., 2003) (Norton et al., 2001; Thimothe et al., 2004) (D’Agostino et al., 2004) (Silbernagel et al., 2004)

BAXR PCR

Competitive PCR

hlyA None hlyA 15 h PCR with DNA probe Commercial probe 16 h

5h 20 h 24 h

FRET–PCR Real Time PCR

hlyA hlyA hlyA, iap

None None None

2.5 h 2.5 h 1h

Commercial kit Commercial kit 23S rRNA

24 h None None

30 h 8h 4h

None

N/Ab

102–103 cfu/ml Pure culture 108–1010 cfu/25 g Cabbage 103–105 genome Pure culture copies N/A Pure cultures

1h 2h

54 hc 55 hc

10–15 cfu/ml 3 cfu/g

Multiplex PCR with Microsphere sorting DNA Microarray iap, hly, inlB, plcA, plcB, clpE RT–PCR iap iap a b c

Pure culture Ground beef

(Choi and Hong, 2003) (Choi and Hong, 2003) (Ingianni et al., 2001) (Koo and Jaykus, 2003) (Koo and Jaykus, 2003) (Rodriguez-La´zaro et al., 2004a,b) (Bhagwat, 2003) (Hough et al., 2002) (Dunbar et al., 2003) (Volokhov et al., 2002) (Klein and Juneja, 1997) (Klein and Juneja, 1997)

The gene amplified is not mentioned for the kit. N/A — not addressed in paper. The long time period includes Southern hybridization to detect the RT-PCR product.

for L. monocytogenes. The only difference is that the PCR method requires only 58–60 h to perform rather than 5 days (Wan et al., 2003). D’Agostino et al. (2004) also developed a PCR assay that incorporated an internal control to make it reproducible between laboratories, but the sensitivity was low — only 89.4% of samples containing 20–200 L. monocytogenes cells/25 g were correctly identified (D’Agostino et al., 2004). Jung et al. (2003) used PCR to detect L. monocytogenes using the internalin AB (inlAB) gene. PCR allowed the specific detection of a number of serotypes of L. monocytogenes while no bands from other non-pathogenic species of Listeria were detected. The limit of detection of the pathogen in pure cultures was 105 cells/ml culture. When frankfurters were spiked with the pathogen, the limit of detection was improved to 10 cells in a 25 g sample, provided the sample was first enriched for at least 16 h (Jung et al., 2003). This method allowed detection of the pathogen within 24 h.

One of the methods becoming popular now, and being recommended in the United States for Official First Action for detection of L. monocytogenes in dairy products, fruits and vegetables (except radishes), seafoods, raw and processed meats and poultry is the BAXR system (Dupont Qualicon, DE) (Silbernagel et al., 2004). In this system, PCR is used to amplify a Listeria-specific DNA fragment. A fluorescent probe in the BAXR mix binds to the double stranded DNA. After PCR amplification, the system heats up the sample and analyses the decrease in fluorescence as the double stranded DNA melts. Analysis of the melting curve allows one to determine whether L. monocytogenes is present in the sample (Silbernagel et al., 2004). Three days of culture enrichment are required before the PCR is run, with the first enrichment medium being specific to the food sample being tested (Silbernagel et al., 2004). Traditional PCR methods are able to detect the presence of the pathogen, but they are not able to

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quantify the level of pathogen contamination. One way to approach this problem is the use of competitive PCR. In this method, a competitor fragment of DNA that matches the gene to be amplified is introduced into the sample. In general, the competitor fragment is synthesized as a deletion mutant that can be amplified by the same primers being used to amplify the target DNA. The competitor fragment is distinguished from the pathogen gene fragment by its smaller size. In order to determine the level of pathogen contamination, DNA purified from the food sample is serially diluted and added to a constant amount of competitor DNA. PCR is performed and the intensity of the pathogen gene’s signal is compared to that of the competitor DNA on an agarose gel. The number of cells in the original sample can be estimated by the intensity of the fulllength PCR product (from the pathogen) as compared to the intensity of the smaller, competitive PCR product (Schleiss et al., 2003) using a standard curve. The advantage of this method over a number of other PCR methods is that no expensive fluorophores or radioactive labels are required to visualize the results (Choi and Hong, 2003). Choi and Hong (2003) used a variation of competitive PCR, based on the presence of a restriction endonuclease site in the amplified gene for L. monocytogenes detection. The competitor fragment was mutated so that the restriction endonuclease site was removed; after PCR and digestion with the endonuclease, the competitor amplicon was visualised as a slightly larger molecule. According to Choi and Hong (2003), the method took around 5 h to complete without enrichment and was able to detect 103 cfu/ 0.5 ml milk using the hlyA gene as the target. The detection limit could be reduced to 1 cfu if culture enrichment for 15 h was conducted first. PCR-based methods have several limitations. First, the food matrix, which includes the fat and proteins in food samples, may interfere with PCR by inhibiting DNA polymerase directly or indirectly by binding Mg2+, as the Mg2+ is required for activity of the Taq polymerase (Fratamico and Strobaugh, 1998; O’Connor, 2003). The matrix-based interference necessitates the use of DNA purification steps that add to both the cost and completion time of the method. Second, culture enrichment may be required to concentrate the pathogen to improve the sensitivity of detecting the target gene. Third, analysis on an agarose gel is labour intensive when done on the large scale required by the food processing industry. Fourth, PCR only detects the presence of DNA. This does not indicate whether the pathogens are dead or alive (O’Connor, 2003). The

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food industry must know whether the food represents a health hazard, not whether the pathogen was present at one point but was killed by the food processing method. Several groups have developed alternatives to the use of classical PCR to enhance pathogen detection. One method used is to combine PCR with the use of oligonucleotide probes. The PCR is performed first, and then oligonucleotide probes complementary to the gene of interest are used to capture and detect only the DNA of interest. Ingianni et al. (2001) were able to detect L. monocytogenes in food samples without enrichment using complementary probes, but admitted that their results were much more reliable after overnight culture enrichment in selective media. Culture enrichment increased their detection time from one working day to two with a detection limit of 2–10 cells/g sample. One rate-limiting step for PCR methods is the need to analyze and detect the amplified DNA product. When dealing with hundreds or thousands of samples, the time for running agarose gels with the DNA samples, or performing DNA hybridization assays becomes a rate limiting exercise (Koo and Jaykus, 2003). Some groups have eliminated the need for running agarose gels by using fluorescent resonance energy transfer (FRET)-based PCR (Koo and Jaykus, 2003). In this method, the DNA product is analyzed directly after PCR by measuring the fluorescence signal (See Fig. 1). This system works by having two DNA probes for the gene of interest, one with a fluorescein label and the other with a quencher label. During the annealing and primer extension steps of the PCR, the fluorescein-labelled oligonucleotide hybridizes to the gene of interest. The hybridized probe is digested by the exonuclease activity of the DNA polymerase as the polymerase amplifies the gene. This digestion releases the fluorophore from the probe. The probe with the quencher label is short and will not anneal to the fluorescein-labelled probe until after the PCR is completed and the mixture is cooled to room temperature. At this point, unused fluorescein-labelled probe is quenched due to its hybridization with the quencher probe, leaving only the free fluorophore (Koo and Jaykus, 2003). The resulting fluorescence, due to free fluorophore, is proportional to the number of pathogens in the original sample and obviates the need to run agarose gels for PCR product detection. A single probe containing both the fluorophore and quencher can also be used (Cox et al., 1998), but the double label significantly increases the cost of probe synthesis. This method, targeting hlyA, provides a detection limit for L. mono-

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Fig. 1. Use of FRET-based PCR to detect pathogens. This method has several components. A) Template DNA from the sample, containing a pathogen-specific target gene is isolated, and oligonucleotide primers specific to the target gene are added to amplify the target gene. A probe specific for a central sequence in the target gene is labelled with a fluorogen and added to the mixture. A short oligonucleotide probe, specific for the fluorescent probe, labelled with a quenching molecule, is also present. This probe has a low melting temperature so that it will not bind to the fluorescent probe during PCR. The PCR mix must contain a DNA polymerase that retains 5V-exonuclease activity. Finally, dNTPs are added to allow the amplification to occur. B) In the first two steps of the PCR, template DNA from the sample is heated to a temperature that will denature the DNA. The temperature is then lowered to allow annealing of the primers for both the amplification and fluorescent probes. C) The pathogen-specific target gene is amplified by Taq DNA polymerase. Any fluorescent probe that has annealed to the DNA is digested by the exonuclease activity of the Taq polymerase to release free fluorogen. D) After a number of rounds of amplification, the PCR products are cooled. At this point, the quencher probe will bind to any fluorescent probe remaining on the template and this prevents fluorescence from being emitted by that probe. The more pathogens initially present in the sample, the more copies of the amplified gene will be present. This leads to more fluorescent probe binding to the gene, and hence being digested by DNA polymerase to release the fluorogen. Only the free fluorogen will fluoresce. At the end of the reaction, the number of pathogen cells in the original sample is estimated from the fluorescence signal; the more pathogen template DNA present initially, the higher the fluorescence. F, fluorescent label; Q, quencher.

cytogenes of about 500 cfu/ml in pure culture, and 103–104 cfu/25 g of inoculated skim milk. The endpoint detection time, after DNA purification, is about 2.5 h (Koo and Jaykus, 2003).

Another way to eliminate the need for agarose gel electrophoresis is to use real-time PCR in a 96-well PCR format. In this method a fluorescent dye, such as SYBR Green I, is used to follow the PCR amplifica-

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tion in real-time and can be used to detect the amplified products from a number of genes at the same time (Bhagwat, 2003). The sample is incubated in a universal enrichment broth, allowing any pathogen present to grow. The DNA from all of the pathogens is isolated and amplified using primers specific to different genes within each pathogen. SYBR Green has an emission spectrum that is 50- to 100-fold brighter when the dye is bound to double stranded DNA. As the DNA is amplified, the increase in fluorescence can be followed as the genes are amplified. Because each gene amplified has a different length and GC content, each gene has a different melting point. Thus a melting curve analysis can be completed post PCR, using the SYBR Green I as a fluorescent marker to measure the melting points. As the melting point is reached, the DNA denatures and the fluorescence decreases sharply. The fluorescence versus temperature graph is plotted to calculate the melting temperature for each product. Each pathogen-specific amplicon has a different melting point so the results of the melting curve analysis can be compared to standards to determine which pathogens are present in the sample. The fluorescence is measured on a plate reader, obviating the need for agarose gel electrophoresis (Bhagwat, 2003). Using real-time PCR, Bhagwat (2003) was able to simultaneously detect L. monocytogenes, S. typhimurium, and E. coli O157:H7 from produce. Including time for culture enrichment, the entire process took around 30 h, and allowed detection limits of 1 cell/ml for E. coli O157:H7 and Salmonella spp. and 102–103 cells/ ml for L. monocytogenes. Wang et al. (2004) were able to reduce this time to 10 h with levels of detection as low as 3 and 4 cfu/g in raw sausage for E. coli O157:H7 and L. monocytogenes, respectively, by reducing the enrichment time and altering the DNA extraction method of the sample. Hough et al. (2002) were able to detect between 103 and 1010 cfu for L. monocytogenes in 25 g samples of cabbage without any culture enrichment. Because of the lack of enrichment, the entire process from extraction to real-time PCR results was around 8 h (Hough et al., 2002). One advantage to using real-time PCR is that primers can be designed to simultaneously detect both Listeria sp. and L. monocytogenes by amplifying the 23S rRNA gene (conserved in all Listeria sp.) at the same time as the hlyA gene (Rodriguez-La´zaro et al., 2004a). The other advantage is that it can be used with the proper primers to quantify the number of pathogens present in a sample by measuring the level of fluorescence as compared to a standard.

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Multiplex PCR is a variation of the traditional PCR. This method makes use of multiple sets of primers to amplify a number of genes or gene fragments simultaneously. For instance, Bhagwat (2003) artificially contaminated produce, cultured the pathogens, then used multiplex PCR to simultaneously detect L. monocytogenes, S. typhimurium, and E. coli O157:H7. This identification procedure is effective, but not conducive to high-throughput screening because of the need to analyze the PCR products by agarose gel electrophoresis. As with real-time PCR, multiplex PCR can also be used with the primers complementary to the highly conserved Listeria 23S rRNA gene and L. monocytogenes specific hlyA gene to simultaneously identify both Listeria sp. and L. monocytogenes in samples (Kanuganti et al., 2002; Rodriguez-La´zaro et al., 2004a). Kanuganti et al. (2002) used this method to identify levels of contamination by Listeria sp. and L. monocytogenes in fresh and processed pork. Numbers of bacteria required for detection were not addressed. In a more elaborate variation of multiplex PCR, Dunbar et al. (2003) amplified portions of the prokaryotic 23S rRNA using universal primers labelled with biotin. After amplification, the PCR product was mixed with fluorescently labelled microspheres displaying pathogen-specific oligonucleotides (See Fig. 2). Each type of microsphere has a different spectral pattern, and each type displays a DNA probe specific for a different pathogen. The microspheres are added to the mixture containing the denatured pathogenic DNA (derived from the PCR amplification), and the DNA will bind to microspheres displaying the complementary DNA probe. Streptavidin-R-phycoerythrin is added, which binds to the biotin label on the amplified DNA. The phycoerythrin fluoresces at a different wavelength than the microspheres. The bound microspheres are sorted according to their spectral signatures, and in the process sort the various pathogen DNA fragments (Dunbar et al., 2003). The amount of fluorescence from the phycoerythrin can then be measured after sorting to determine the quantity of pathogen DNA present in each species. Theoretically, this system could be used to simultaneously detect hundreds of different pathogens. Using the Luminex LabMAP (Luminex, Austin TX) system, Dunbar et al. (2003) detected L. monocytogenes, E. coli, Salmonella and C. jejuni simultaneously at levels of 103 to 105 organisms. The main advantage of this system is that after enrichment and DNA amplification, enumeration of the number of pathogens takes only 30–40 min (Dunbar et al., 2003) and multiple pathogens are detected simultaneously. However, this system is prohibitively expensive, still requires

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Fig. 2. Multiplex PCR and the use of fluorescently labelled microspheres to detect pathogens. A) DNA is isolated from the food sample and all prokaryotic 23S rRNA gene sequences are amplified using biotinylated universal primers, resulting in the addition of a biotin label to the amplified DNA. Fluorescently labelled beads containing a different spectral pattern for each pathogen-specific probe are added to the amplified PCR mix, and the DNA denatured to create single-stranded fragments. B) The amplified pathogenic DNA fragments bind to the beads displaying their complementary probes. C) The bound microspheres are labelled with a fluorescent label by adding streptavidin-R-phycoerythrin, which binds to the biotin label on the amplified DNA and fluoresces at a wavelength different from that of the microspheres. D) The beads bound to the pathogenic DNA are sorted based on their spectral patterns, and the amount of fluorescence measured to quantify the amount of each pathogen present. B, biotin; P, phycoerythrin.

time for culturing, and may experience difficulties with matrix effects. To address the need of detecting only living pathogens, one can detect the pathogen RNA rather than DNA. The presence of specific RNA sequences is an indication of live cells. When an organism dies, its RNA is quickly eliminated, whereas the DNA can last for years, depending on storage conditions. Reverse transcription-PCR (RT-PCR) makes use of a reverse transcriptase that is able, in the presence of a complementary primer, to

create complementary DNA (cDNA) from an RNA strand corresponding to a transcribed gene. The cDNA is then amplified using oligonucleotide primers and DNA polymerase under normal PCR conditions. Analysis of the results is done in the same way as for PCR. Klein and Juneja (1997) used RT-PCR to detect live L. monocytogenes in pure culture and artificially contaminated cooked ground beef. In pure culture, the authors used culture enrichment for one h, followed by isolation of total RNA from the pathogen. They

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performed RT-PCR on the iap, hlyA and prfA genes. The authors visualized the specific RT-PCR product using Southern hybridization with a probe specific for the gene. Using the iap gene as the target, the authors were able to detect 10–15 L. monocytogenes cells/ml in pure culture. RT-PCR with the other two genes was not as sensitive, requiring either a higher number of organisms or more enrichment time. When the authors used RT-PCR on the iap gene to detect the pathogen in ground beef, they detected organisms at a level of 3 cfu/g of inoculated meat after 2 h of culture enrichment. Total time for detection using this method was 54 h (Klein and Juneja, 1997). Rudi et al. (2005) added ethidium monoazide bromide (EMA) to their samples prior to using PCR for detection. This stain selectively enters cells with damaged membranes, and binds to DNA. DNA bound to the EMA cannot be amplified by PCR, and thus only the DNA of live cells is amplified by PCR, and only this DNA will be detected. The authors were able to detect about 102 cfu/g in gouda-like cheeses. This technique also is able to separate viable-but-not-culturable cells from dead cells (Rudi et al., 2005). 2.4.2. NASBA — nucleic acid sequence-based amplification Beumer and Hazeleger (2003) used in vitro RNA amplification to detect viable pathogens since RNA is unstable and likely to be present only in living cells. In nucleic acid sequence-based amplification (NASBA), total RNA in a sample is extracted. Messenger RNA is then used as the target because it predicts viability better than rRNA or total RNA, and the PCR amplification step is not used so that no thermocycler is required. The following three enzymes are used during NASBA: reverse transcriptase, RNaseH, and T7 RNA polymerase. Two oligonucleotide primers specific to the gene of interest are used, and a mixture of both NTPs and dNTPs are added. Primer 1 (P1) is complementary to the mRNA and contains a T7 promoter sequence. It binds the mRNA, and the reverse transcriptase produces a cDNA molecule. RNaseH digests the RNA in the RNA:DNA hybrid. Primer 2 (P2) can then bind to the cDNA and the DNA-dependent DNA polymerase activity of the reverse transcriptase allows it to synthesize the complementary strand (Kievits et al., 1991). This creates a bminigeneQ which is transcribed into thousands of RNA transcripts by the T7 RNA polymerase (Cook, 2003). Each of the RNA transcripts can be recognized by P2, thus creating a cycle of amplification where the main product is the RNA transcript. The transcripts can be detected by agarose gel

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electrophoresis or by probe hybridization. Because the reaction is done at 41 8C, the genomic DNA stays in its double-stranded form and is not a target for amplification from the reverse transcriptase (Cook, 2003). For more details see Fig. 3. NASBA has been used to detect viable L. monocytogenes at 10 cfu/60 g meat or seafood product (Blais et al., 1997; Uyttendaele et al., 1995). The pathogen was detected using the 16S rRNA sequences and also the hlyA mRNA (Blais et al., 1997). The assay took three days to perform, including culture enrichment (Uyttendaele et al., 1995). Aside from ensuring detection of only viable cells, NASBA removes the need for a costly thermocycler. NASBA still requires that nucleic acids be extracted from the food samples, so there may be matrix effects on the enzymes (Cook, 2003). 2.4.3. DNA microarrays Microarrays are composed of a number of discreetly located DNA probes fixed on a solid substrate such as glass. Each probe corresponds to an oligonucleotide specific to a target DNA sequence. Call et al. (2003) used probes specific for unique portions of the 16S rRNA gene in Listeria sp. to demonstrate how each Listeria species could be differentiated by this method. In this procedure, PCR is first performed using universal primers to amplify all the 16S rRNA genes present in a sample. The various amplified DNA fragments bind only to the probes for which they have a complementary sequence. Because one of the oligonucleotides used in the PCR contains a fluorescent label, the spots where the amplified DNA has bound fluoresce. Pathogens are identified by the pattern of fluorescing spots in the array. Alternatively, multiple primer sets can be used simultaneously to amplify a number of pathogen-specific genes in a multiplex PCR, for example using iap, hlyA, inlB, plcA, plcB, and clpE (Volokhov et al., 2002). Multiple primer sets can be used in the PCR because only those products binding to a microarray probe will be detected so one is not limited by the number of resolvable bands on an agarose gel (Call et al., 2003). Sergeev et al. (2004) developed a microarray that was capable of detecting four Campylobacter species of clinical relevance, six Listeria species, 16 staphylococcal enterotoxins, and six C. perfringens toxins. Based on previous experience, the authors claim that in pure culture the detection limit of the array is 200 cfu L. monocytogenes (Lampel et al., 2000; Sergeev et al., 2004). They also claim that the array is appropriate for the detection of the pathogens in food and environmental samples (Sergeev et al., 2004). Microarrays are able

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Fig. 3. A schematic representation of NASBA. In NASBA, three enzymes are required: a reverse transcriptase (RT) containing DNA-dependent DNA polymerase activity, RNaseH, and T7 polymerase. NTPs and dNTPs are also included in the mixture. Primer 1 (P1) contains a T7 promoter and is complementary to the pathogen-specific mRNA. A) P1 binds the mRNA and the RT extends the primer, creating a cDNA molecule complementary to the mRNA. B) RNaseH degrades the RNA in RNA:DNA hybrid. C) Primer 2 (P2) binds to the cDNA and the RT extends the strand due to its DNA-dependent DNA polymerase function. D) This bminigeneQ is then transcribed in great numbers by the T7 polymerase in the mix. E) The transcribed minigene will bind to P2 that starts a cycle resulting in large amounts of the minigene being synthesized. This product can be analyzed by agarose gel electrophoresis or by various blotting techniques. The entire amplification is done at 41 8C so that the genomic DNA stays in its double stranded form and is not available as a target for the RT. Dark wavy lines represent the native mRNA; light wavy lines represent RNA synthesized from the minigene; straight lines represent DNA; dotted lines are degraded RNA. Adapted from (Kievits et al., 1991).

to identify a number of pathogens or serotypes at once, but they still require culture enrichment and PCR steps to improve the sensitivity and specificity of detection. 2.5. Pathogen subtyping and verification methods RFLP (restriction fragment length polymorphism), RAPD (randomly amplified polymorphic DNA), AFLP

(amplified fragment length polymorphism), ribotyping and PFGE (pulsed-field gel electrophoresis) are molecular-based methods that are used for further verification after a food sample is suspected of harbouring a pathogen. These methods are not used as routine screening tools. Each allows further characterization of the pathogen to determine which of the many possible subtypes within a serotype is causing the disease

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or is the contaminating factor. The information is important from epidemiological and source-tracking points of view and is needed to identify the precise strains responsible for causing disease, or that contaminate a certain type of food. The main drawback with these methods is that individual cultures have to be isolated and grown before being analysed independently. The methods are compared in Table 3. 2.5.1. RFLP — restriction fragment length polymorphism Restriction fragment length polymorphism (RFLP) analysis uses the restriction endonuclease patterns in DNA to determine differences in genetic profiles. Even among closely related individuals, the mutation of a restriction sites occurs in sufficient frequency to allow a pedigree to be formed (Smith and Nelson, 1999). Digesting genomic DNA using restriction endonucleases creates fragments of different lengths. The

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DNA is then run on a capillary electrophoresis apparatus and the banding patterns analyzed. Different groups of microorganisms, or serotypes within the same species can be detected from the differences in the electrophoretic patterns of DNA fragments. However, digesting genomic DNA often results in too many bands, leading to difficulty in interpretation of results. To minimize interference, RFLP can be done on individual genes. Paillard et al. (2003) used PCR– RFLP on the 23S rRNA gene to determine the species of Listeria in sludge samples. They first enriched all Listeria species from the sludge, and plated them. Presumptive Listeria colonies were grown in pure culture and subjected to RFLP to identify the Listeria species. While they were able to determine the species with great accuracy, the requirement for using pure cultures makes the method time consuming. Weidmann et al. (1997) used RFLP on the hlyA, actA and inlA genes of L. monocytogenes to determine how strains

Table 3 Comparison of pathogen subtyping and verification methods Methods

Advantages

Disadvantages

Amount of DNA required

AFLP

–PCR to amplify a few genes and simplify analysis of results –Can compare differences between bands on a gel –Fewer bands to compare than with RFLP –Can compare among labs –May detect one pathogen in a mixed community of microbes –PulseNet database available to coordinate and compare results — can track outbreaks –Can compare differences between bands on a gel –Results are comparable among labs

–Requires pure cultures of pathogen –Cannot compare organisms at the genus or family level

10–100 ng

–Cells must be fixed and permeabilized before visualization –Time consuming 3 days for results –Requires pure cultures of pathogen –Patterns may change after intestinal passage; differences in patterns may not indicate actual strain differences –Cannot compare organisms at the genus or family level –Requires pure cultures of pathogen –Is not very reproducible between gels or labs –Cannot compare organisms at the genus or family level –Requires pure cultures of pathogen –Many bands to compare — confusing, may be hard to see differences –Cannot compare organisms at the genus or family level –Partial digestion and faint bands may be a problem –Requires pure cultures of pathogen

Single cell level

FISH PFGE

RAPD

–Arbitrary primers that are not gene specific

RFLP

–Simplicity –Can be done with restriction endonucleases for serotype level discrimination or combined with PCR for lineage comparisons

Ribotyping

–Similar to RFLP, but visualises only bands corresponding to the rrn portion of ribosomes so fewer bands to compare –Can compare organisms at the genus and family level

Adapted from (Gurtler and Mayall, 2001; Savelkoul et al., 1999).

Measured by turbidity of culture — lysis and DNA digestion performed in agarose plugs

10 ng

3–5 Ag

1 Ag

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with different pathogenic potential were related. They found that L. monocytogenes could be divided into three lineages, and that all epidemic outbreaks could be traced back to lineage I. RFLP has been modified in recent years to provide data that is more comparable between labs. Two of the methods developed are ribotyping and pulse-field gel electrophoresis. These techniques are outlined below. 2.5.2. Ribotyping Ribotyping is similar to RFLP in that it uses restriction endonuclease digestion of DNA to create a pattern that can be analysed. This technique relies specifically on the ribosome encoding genes that are relatively conserved across the bacterial kingdom, and allows lineages to be traced through the appearance of mutations over time. To perform the procedure, genomic DNA from individual strains is first digested with an endonuclease such as EcoRI, followed by Southern hybridization with a probe specific to conserved regions of the rRNA coding sequence. The probe allows only the DNA fragments encoding rRNA to be detected on the blot (Ryser et al., 1996; Weidmann et al., 1997). Different strains will give rise to different banding patterns that are used to determine lineages. Weidmann et al. (1997) used ribotyping on different isolates of L. monocytogenes to determine how different lineages could be grouped and how the groups were related to the pathogenicity of the organism. The authors digested the bacterial DNA using EcoRI and probed with the E. coli rrnB rRNA operon probe, since the gene is conserved across the Eubacteria kingdom. The ribotyping results divided the 133 L. monocytogenes strains into 23 different patterns. The authors then combined this information with analysis of polymorphism in the virulence genes hlyA, inlA, and actA to show that the strains could be divided into three distinct lineages. Of the three, only one (hlyA) was able to separate the lineages into groups correlating to strains causing listeriosis (Weidmann et al., 1997). Suihko et al. (2002) demonstrated the use of ribotyping to track the geographical versus food type prevalence of various ribotypes of L. monocytogenes. The researchers first enriched 564 L. monocytogenes isolates from six meat, two poultry and five seafood plants in the Faroe Islands of Finland using enrichment culturing, and then performed automated EcoRI ribotyping. The group demonstrated that there were geographical differences in the ribotypes, but not differences in the ribotypes of L. monocytogenes in the different types of food being processed.

Manfreda et al. (2005) used EcoRI ribotyping to demonstrate that there is a low level of genetic diversity among L. monocytogenes contamination of Gorgonzala cheeses, with 70% of the contaminating isolates falling into the same ribotype. The data also showed that 90% of the contaminating isolates were associated with type II pathogenicity lineage, which contributes to 35% of the sporadic listeriosis cases. Mereghetti et al. (2002) analyzed the type II lineage using ribotyping and random multiprimer DNA analysis to show that it is more heterogeneous than the type I lineage, and probably diverged from the common ancestor earlier than the isolates of lineage I. 2.5.3. PFGE — pulsed-field gel electrophoresis Pulsed-field gel electrophoresis (PFGE) allows for the separation of large fragments of DNA, from 10– 2000 kb (Finney, 2000). DNA fragments have an overall negative charge proportional to their sizes, due to the phosphate moiety on each nucleotide. When an electric current is applied to a DNA sample in a gel, the DNA moves towards the positive electrode (anode). Because the matrix of the gel acts as a sieve, larger pieces of DNA are retarded in their movement, while smaller fragments travel through the spaces more easily and migrate further on the gel. In a normal agarose gel, the largest size of DNA that can be effectively separated is between 20 and 40 kb. Above this size, DNA fragments must mould into a shape that can squeeze through the sieve; all the large fragments then migrate at around the same rate. PFGE takes advantage of the time it takes for the large DNA fragments to squeeze into their elongated shapes for movement, as a basis for further separation. Larger DNA fragments take longer to be forced into these shapes. Therefore, if the direction of the electric field is changed, then the smaller pieces of DNA will alter their shape faster and start to migrate at the limiting mobility rate. By optimizing the times and angles of this alternating electrophoretic field, larger pieces of DNA can be resolved on the gel (Finney, 2000; Moore and Datta, 1994). In order to perform PFGE, genomic DNA is digested using an infrequently cutting endonuclease and the fragments run on a gel (Yde and Genicot, 2004). Within L. monocytogenes, the DNA is generally cut using AscI, ApaI or SmaI and the patterns are compared to determine the relatedness of the L. monocytogenes strain (Miettinen et al., 1999; Moore and Datta, 1994). Moore and Datta (1994) used SmaI to digest DNA from L. monocytogenes isolated from two listeriosis

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outbreaks in Nova Scotia and Los Angeles. They found that the PFGE patterns from within each outbreak were much more closely associated in terms of banding patterns than those between the outbreaks. This contrasted with previous data from serotyping that suggested the Los Angeles and Nova Scotia outbreaks might be clonal since they shared the same serotype. Vogel et al. (2004) also compared PFGE typing of L. monocytogenes to serotyping and found that after digestion with ApaI, PFGE was more discriminatory at sorting strains of L. monocytogenes into different groups than serotyping. PFGE was able to sort 96 strains of L. monocytogenes into 39 groups while serotyping could only divide the same strains into 2 groups. Aarnisalo et al. (2003) showed that PFGE was slightly more discriminating than ribotyping, separating 486 L. monocytogenes isolates from 17 Finnish food processing plants into 46 PFGE types over 23 ribotypes. The US Centre for Disease Control (CDC) initiated a collaborative effort with many public laboratories to create an electronic database known as PulseNet, which catalogs the PFGE patterns of E. coli O157:H7, non-typhoidal Salmonella serotypes, L. monocytogenes and Shigella (Swaminthan et al., 2001). As of 2002, 46 state labs, two public labs, the Food and Drugs Administration (FDA), US Department of Agriculture (USDA), and several Canadian labs have entered data into the PulseNet. This has allowed for better tracking and earlier detection of possible common source outbreaks (Kathariou, 2002; Swaminthan et al., 2001). Although this method is finding increased use and provides a great deal of information, it is timeconsuming, requiring about three days to obtain results (Finney, 2000). 2.5.4. AFLP — amplified fragment length polymorphism Amplified fragment length polymorphism (AFLP) analysis is a DNA fingerprinting technique based on the selective amplification of genomic restriction fragments to generate a restriction pattern of the amplified bands (Aarts et al., 1999). It essentially combines the reliability of RFLP with the power of PCR. Genomic DNA is digested with two restriction endonucleases, one of which cuts infrequently, and the other on a frequent basis (Blears et al., 1998). Selective pressure for amplification is then applied in two different ways. In the first way, adapter oligonucleotides are ligated to the digested DNA. The adapters add a few selective nucleotides after the restriction site. Only those fragments with the complementary sequence between the

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restriction site and the adaptor will be bound and amplified by PCR. This step results in about 1 / 16 of the bands being amplified during the PCR (Aarts et al., 1999). Selective amplification results in 50 to 100 different fragments being amplified (Blears et al., 1998; Vos et al., 1995). Most of the time, [g 33P]ATP is added to the end of the primers used in the amplification process and the amplified fragments viewed by autoradiography (Vos et al., 1995) or through the use of fluorescent labels (Aarts et al., 1999). If the fluorescent detection method is used, the fingerprinting pattern can be viewed by automated laser fluorescence analysis, which allows comparison of data from other laboratories (Aarts et al., 1999). Genetic polymorphisms are then identified by the presence or absence of different fragments (Blears et al., 1998). AFLP can be used to differentiate strains of L. monocytogenes on a more discriminating basis than serotyping (Aarts et al., 1999; Vogel et al., 2004). Vogel et al. (2004) differentiated between 96 different strains of L. monocytogenes using AFLP. They digested the bacterial DNA with BamHI and EcoRI, before performing AFLP, then used fluorescent labelling to detect the amplified DNA fragments. The authors compared the AFLP results to results obtained by classifying the same strains by RAPD, PFGE, ribotyping, serotyping and PCR–RFLP using the hlyA gene. AFLP grouped the L. monocytogenes strains into 45 types, with similar results being obtained with PFGE and RAPD. Ribotyping was not as discriminatory, dividing the strains into only 16 types, while serotyping had the least discriminatory power, dividing the 96 strains into 2 groups (Vogel et al., 2004). The results demonstrated that AFLP is as effective as RAPD and PFGE for differentiating between L. monocytogenes strains and is less time consuming than PFGE. 2.5.5. RAPD — randomly amplified polymorphic DNA Randomly amplified polymorphic DNA (RAPD) analysis makes use of a short arbitrary primer (e.g. 10 bp) that anneals randomly along genomic DNA to amplify a number of fragments within the genome. As long as the same primer is used for all the test samples, the comparison of the number and sizes of fragments generated allows for discrimination between strains of a pathogen. It does require having a pure culture so that there are no contaminating bands from other organisms or from the DNA in the food sample (Lawrence and Gilmour, 1995). The technique can be used to trace the source of L. monocytogenes contamination in food processing plants. The technique was used to follow the incidence and typing of L. mono-

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cytogenes in both poultry (Lawrence and Gilmour, 1995), and vegetable (Aguado et al., 2004) processing plants. By taking samples throughout the year, culturing the isolates, and performing RAPD analysis on individual isolates, Lawrence and Gilmour (1995) showed that two strains of L. monocytogenes were present in a poultry processing plant throughout the year-long study. They showed that the strains were persistent, and that they might have been responsible for crosscontamination of the preparation areas within that environment. Furthermore, the researchers were able to pinpoint the source of the contamination by these strains. These authors also demonstrated that other strains isolated over time were transient in nature, and probably came from various sources of contamination (Lawrence and Gilmour, 1995). This method is good for microbial source tracking and determining critical control points for preventing pathogen contamination within the food processing industry. 2.5.6. FISH — fluorescence in situ hybridization Fluorescence in situ hybridization (FISH) is often used to study, in a cultivation-independent way, the presence and distribution of specific strains in microbial communities. FISH can be used in phylogenetic studies, and in assessing the spatial distribution of target microbes in communities such as biofilms (Wagner et al., 1998). The method has only recently been applied to detecting and analyzing microbes in food. To perform FISH, a sample is fixed to a slide, membrane filter or well using ethanol. In the case of Gram-positive organisms such as L. monocytogenes, the fixed cells are permeabilized with proteinase K so that fluorescent probes are able to enter the cell. Specific probes are created that are complementary to pathogen DNA and labelled with a fluorescent tag such as Cy3, Cy5 or CFLUOS. The fixed cells are incubated with the labelled probes that will bind complementary sequences. The labelled cells can then be visualized with a fluorescence microscope (Ootsubo et al., 2003; Schmid et al., 2003; Wagner et al., 1998) (See Fig. 4). Wagner et al. (1998) enriched Listeria cells from milk samples in broth for 1, 2 or 7 days before analysis. Using labelled primers specific for the 16S rRNA genes, the researchers were able to detect Listeria reliably on the genus level, allowing for further analysis by competitive PCR. They were also able to perform FISH using oligonucleotide probes specific for the virulence gene transcript, iap-mRNA, which allowed them to analyze virulence gene expression of L. monocytogenes within a mixed microbial community (Wagner et al., 1998).

2.6. Immunoassay-based methods for detection of L. monocytogenes Immunoassays are based on the natural affinity of antibodies for their antigens. The antigen can be a hapten, a protein, or a carbohydrate on the surface of a cell. These assays are fast and relatively inexpensive. They allow accurate detection of antigens after very little sample purification (Hall et al., 1989b). Immunoassays are not as susceptible to matrix effects as PCR assays. For example, samples such as river water can be analyzed directly by ELISA (enzyme-linked immunosorbent assay) (Hall et al., 1989a). In addition, immunoassays can be used to provide real-time information and allow for a timely response if quantities of the pathogen are high enough (Meng and Doyle, 2002). Problems with immunoassay-based methods that may arise are the low sensitivity of the assays, low affinity of the antibody to the pathogen or other analyte being measured, and potential interference from contaminants. Improvements in these areas will likely expand the use of immunoassays in a variety of fields, including the food industry. A comparison between various immunoassay methods to detect foodborne pathogens is presented in Table 4. Most L. monocytogenes detection methods published have used culture-based or nucleic acid-based method. There have been some papers on the use of antibodies for detecting the pathogen, but these are much fewer in number. The most commonly used immunoassays for the detection of the pathogen are based on the use of whole cells. Often L. monocytogenes is enriched from the food sample, heat killed (Beumer and Brinkman, 1989; Comi et al., 1991; Curiale et al., 1994; Gangar et al., 2000; Mattingly et al., 1988; Sewell et al., 2003; Silbernagel et al., 2005), or formalin-fixed (Sølve et al., 2000), and then detected by an ELISA. The problem with whole cell detection is that many of the cell-surface antigens are genus specific rather than L. monocytogenes specific (Durham et al., 1990; Feldstine et al., 1997a,b; Knight et al., 1996). From a foodborne pathogen-detection perspective, this is important since of the six known Listeria species, only L. monocytogenes is pathogenic to humans. In order to become more species specific, some authors have detected the flagella of the bacterium rather than the whole cells (Farber et al., 1988; Kim et al., 2005; Skjerve et al., 1991). These assays require washing in PBS and extraction of the flagella before the assays are done and have often still displayed only genus-specific results (Farber et al., 1988; Kim et al., 2005). ELISAs using the O and H antigens (Palumbo et al., 2003), as

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Fig. 4. A schematic of FISH. In FISH, a sample is first fixed to a slide using ethanol. After drying, Gram-positive cells are permeabilized with proteinase K in order to allow a probe to enter the cell. A solution containing a DNA probe with a fluorescent label specific for the pathogen is incubated with the slide. The probe will bind to its complementary DNA sequence, present only in the species of interest. The slide is washed, and the cells visualized with a fluorescence microscope. Any pathogenic cells will fluoresce due to the presence of the probe.

well as whole cell protein extracts (Bourry et al., 1997) have also been used to detect the pathogen. A few researchers have also used LLO as the detection target (Matar et al., 1992; Paoli et al., 2004). 2.6.1. Types of antibodies used in detection of pathogens A number of antibody types and formats are available for immunodetection. These include conventional and heavy chain antibodies, as well as polyclonal, monoclonal or recombinant antibodies (see Fig. 5). Polyclonal antibodies have been used as detection vehicles for several decades (Breitling and Du¨bel, 1999). These antibodies are raised by immunizing an animal host with the antigen several times, and then harvesting serum from the animal. The serum obtained contains a

mixture of antibodies, most of which do not bind to the antigen and were present before immunization. However, if the antigen is immunogenic and contains a number of epitopes to which antibodies can bind, this is a simple way of obtaining a detection reagent for performing immunoassays. The supply of the polyclonal antibodies only lasts as long as the animal since different animals will have different antibody responses to the antigen challenge. This type of antibody production has been used in a number of commercial assays, including the TECRA Listeria Immunoassay (TLVIA) (Knight et al., 1996) and Assurance Listeria polyclonal enzyme immunoassay (Feldstine et al., 1997a). A detailed description of these assays is included below. Monoclonal antibodies are often more useful for the defined, specific detection of a molecule than polyclon-

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Table 4 Comparison between immunoassay methods to detect L. monocytogenes Method

Advantages

Disadvantages

Detection limit

ELISA

–Simplicity –Detection within a few hours

Sandwich ELISA

–Concentrates the antigen in the well –Gives a stronger signal

103–105 cfu/ml (de Boer and Beumer, 1999); 103–106 cfu/ml (Crowley et al., 1999) 1 cfu/25 g after enrichment (Durham et al., 1990)

Competitive ELISA

–Less false positives –Allows for quantitative results –Less time required to measure signal

–Large quantities of sample required –Background levels can be high –Must have a second primary antibody for a different portion of the antigen than the first primary antibody –More time required than basic ELISA –Must have pure antigen available –More expensive than ELISA –Specialized equipment required to analyze results –Expensive chemicals required –Trained personnel required –Computer sorter is specialized and expensive

Fluorescently labelled immunoassay Fluorescent immunoassay with microsphere sorting

Latex agglutination assay

–30–40 min for detection after enrichment –Sensitive –Multiple pathogens can be detected simultaneously –No skilled help required for assay –Inexpensive –No specialized equipment

al antibodies because they provide an indefinite supply of single antibody. Raising a monoclonal antibody also begins with the immunization of an animal. Once the immune response has been maximized, the B-cells producing the antibodies are isolated from the spleen of the immunized animal and fused with myeloma cells to produce a hybridoma cell that has the immortality of a cancer cell and the antibody producing capability of a B-cell. Monoclonal antibodies have a number of advantages over polyclonal antibodies. First, hybridoma cell lines expressing monoclonal antibodies can be cultured indefinitely in vitro to provide a continuous supply of homogeneous, well-characterized antibodies. Clones can be selected with different specificities and affinities for a molecule, or a family of molecules. Non-specific antibodies are removed during the selection process, thereby limiting interference in the assay (Deschamps and Hall, 1990; Deschamps et al., 1990). Monoclonal antibodies are used in the Vitek Immuno Diagnostic Assay System (VIDAS)-LMO (bioMe´rieux Vitek, Hazeltown, MO) and Lister test (Vicam, Watertown MA); both assays are commercially available (Allerberger, 2003; Sewell et al., 2003). Nato et al. (1991) developed nine monoclonal antibodies to LLO. Six of the antibodies had equilibrium dissociation constants for the toxin in the nanomolar range, varying from 3.0  10 9 to 5.0  10 7 M. Of

–Qualitative results only –Enrichment culturing required

Not determined 104–105 cfu/ml in pure culture (Sewell et al., 2003)

500 cfu/ml (Dunbar et al., 2003)

0.3–222 cfu/g after enrichment (Matar et al., 1997)

these, two cross-reacted with other cholesterol-dependent cytolysins and one recognized seeligerolysin O from L. seeligeri. Three of the antibodies were capable of neutralizing the haemolytic activity of the haemolysin (Nato et al., 1991). The different properties of the monoclonal antibodies allow them to be used for different applications, although only those specific for L. monocytogenes are useful from a pathogen detection point of view. The problem with monoclonal antibodies is that they are expensive to produce, requiring a skillful technician and specialized growth apparatus for tissue culturing. Also, the production levels are generally low. For instance, of the three monoclonal antibodies produced by Nato et al. (1991) that are appropriate for use in L. monocytogenes detection, the highest yield of antibody was 6 Ag/ml. Within the food industry, what is needed is a cheap supply of large amounts of antibodies to perform high-throughput screening for pathogens. Recombinant antibodies fill this void since they can be produced in reasonable quantities in short periods of time from bacterial expression systems. Handling, growth and storage of bacteria is simpler and cheaper than the corresponding work with hybridoma cells (Breitling and Du¨bel, 1999). Recombinant antibodies also have the advantage of being clonal in that one antibody fragment is produced per bacterial colony. The use of recombinant antibodies offers the opportunity to

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Fig. 5. A) Conventional IgG antibody and various possible recombinant fragments derived from the antibody. The Fab is composed of the first constant domain and the variable domains of both the heavy and light chains. The scFv is composed of the variable light and heavy domains connected and stabilized by a flexible peptide linker. The VH is derived from only the heavy chain variable region. B) The camelid heavy chain antibody (HCAb) and its recombinant derivative VHH. The VHH is derived from the variable region of the HCAb.

select binding antibodies from naı¨ve rather than immunized antibody libraries, removing any ethical dilemma of immunizing animal hosts with known pathogenic organisms (Churchill et al., 2002) or sacrificing the animals for their spleens. Churchill et al. (2005) recently expressed recombinant LLO for use in selection of recombinant antibody fragments from a naı¨ve library. Finally, recombinant antibodies can easily allow one to improve the binding properties and affinities of an antibody through mutagenesis and genetic engineering of the expression vectors and genes (Yau et al., 2003). Although they are in development (Paoli et al., 2004), there are as yet no proven anti-Listeria or anti-LLO recombinant antibodies in the literature, but the development of such recombinant antibodies would be highly desirable because of the various advantages of recombinant antibodies listed below. Recombinant antibodies can be made in a number of forms (see Fig. 5). The Fab fragment is made up of the first constant domain and variable domain of both the heavy and the light chains of the antibody and a disulfide bond joins the two chains. Fab fragments have comparable affinities to the monoclonal antibodies from which they are derived. However, their expression by bacteria may be difficult since the fragment is too

large to be expressed intact, and bacteria do not have the machinery to form disulfide bonds correctly in the cytoplasm. Therefore, each chain must be expressed separately and recombined (Churchill et al., 2002; Hudson et al., 2001; Joosten et al., 2003; Little et al., 2000; Yau et al., 2003). To further reduce the size of the expressed fragment, scientists removed the constant domains from the Fab fragments, leaving the heavy and light variable fragment (Fv). These fragments have higher levels of expression in bacterial cells, due to their smaller size, but tend not to combine because the heavy and light chains of the Fv are not attached by a disulfide bond and must be stabilized. Joining the heavy and light chains by short synthetic peptide linker results in the formation of a single chain variable fragment (scFv) (Fig. 5). scFvs have a number of advantages over other constructs. They are well expressed in bacterial systems, and can be expressed as a single gene construct. This eliminates the need for re-association of the heavy and light chains. They have affinities that are generally comparable to the original antibody, but contain only a single binding domain rather than the two found in conventional antibodies. Paoli et al. (2004) recently

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isolated phage-displayed scFvs against whole L. monocytogenes cells. While the paper did not show binding of free scFv to L. monocytogenes or demonstrate binding of the recombinant antibody to the pathogen in pure culture and after isolation from food, it is a start to the use of recombinant antibodies for detecting L. monocytogenes in food samples. More recently, a different type of recombinant fragment has been constructed, called the VHH (the designation VHH distinguishes this heavy chain variable region from the corresponding VH domain of conventional antibodies). Llamas and camels, members of the camelid family, have antibodies that lack a light chain but are stable under biological conditions and have good binding affinities for antigens (Churchill et al., 2002). These antibodies consist of only a heavy chain dimer (Fig. 5) (Hamers-Casterman et al., 1993; Woolven et al., 1999). Within the variable (VHH) domain are several amino acid substitutions in the framework regions as compared to the VH of conventional antibodies. These substitutions stabilize the antibody fragment, increase its binding affinity to antigens, and increase its expression levels, while simultaneously inhibiting any possible interactions with a VL domain (Vu et al., 1997). This allows convenient expression of VHHs as recombinant fragments from bacterial systems. VHHs are smaller than scFvs, and thus can be expressed to a higher level in bacterial systems. They do not contain a linker that sometimes causes aggregation in scFv constructs, and they are stable, remaining active in some cases for more than a year at 48C (unpublished observations). We have recently used naı¨ve VHH-based phage- and ribosome display libraries to isolate antibody fragments against LLO for use in the detection of L. monocytogenes in food (Churchill et al., unpublished). 2.6.2. ELISA — enzyme linked immunosorbent assay ELISAs are the most common format used for immunodetection of pathogens. By this method, most pathogens have a detection limit of between 103 and 105 cfu/ml (de Boer and Beumer, 1999). To achieve this detection limit often requires enrichment of the pathogens for at least 16–24 h before the concentration of the pathogen is adequate for detection by ELISA (de Boer and Beumer, 1999). There are a number of ELISA formats including direct ELISAs, sandwich ELISAs and competitive ELISAs. In a direct ELISA, the test sample is coated onto a well in a microtitre plate. The plate is blocked and washed, and then an antibody (primary antibody) specific for the pathogen is added to the well. After a

suitable incubation period, the plate is washed to remove unbound antibodies. A secondary antibody conjugated to an enzyme that converts a colourless substrate to a visible product is added. The secondary antibody has specificity for the primary antibody, so if the primary antibody is bound to any antigen, the secondary antibody will bind to the primary antibody. Finally, the plate is washed again to remove any excess secondary antibody, and a substrate is added that turns colour in the presence of the enzyme. The intensity of the colour is proportional to the amount of pathogen present in the original sample (Hess et al., 1998). Some examples of direct ELISAs performed in recent years include a phage ELISA against L. monocytogenes using an scFv antibody fragment (Paoli et al., 2004), which provides one of the few examples of an antibody against whole cells that is L. monocytogenes specific. The limit of detection was not determined as the affinity of the scFv was too low. Palumbo et al. (2003) used commercial antibodies against the H and O antigens of L. monocytogenes to detect the pathogen in a direct ELISA format against immobilized colonies. The authors were able to detect individual L. monocytogenes colonies from a blotted enrichment plate. However, most of the antibodies used in direct ELISAs to date have been only genus specific. Lathrop et al. (2003) characterized a monoclonal antibody that was able to detect heat-killed Listeria sp. The antibody was also specific to the 76, 66, 56 and 52 kDa proteins from L. monocytogenes and the 66, 56, and 52 kDa proteins from L. innocua, making it too non-specific for a foodborne detection system. The antibody was unable to detect viable cells at levels greater than 108 cfu/ml. Farber et al. (1988) used an anti-flagellar antibody to detect both flagella in a well and immobilized Listeria sp. colonies from a plate. The detection limit of the method was not addressed and the antibody was only specific to a genus level. In a sandwich ELISA, the plate can first be coated with an antibody specific for the pathogen. The sample is then incubated in the well, and the target antigen binds to the bound antibody. After washing away the unbound sample, a second antibody with affinity for the antigen is added. The rest of the sandwich ELISA follows the same protocol as for a conventional ELISA. The modification allows concentration of the sample, increased intensity of the signal, and a lower limit of detection (Hess et al., 1998). There are a number of papers that have utilized the sandwich ELISA approach. Both Farber et al. (1988) and Kim et al. (2005) made use of monoclonal antibodies against L. monocytogenes flagella. Kim et al. (2005)

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were able to reduce their detection limit to 105 cfu/ml using two different anti-flagellar monoclonal antibodies, but the detection was still only specific to the genus level. The sandwich ELISA format has also been used in the TECRA Listeria Visual Immunoassay (TLVIA) (Knight et al., 1996) and the Assurance Listeria polyclonal enzyme immunoassay (Feldstine et al., 1997a). Both assays had comparable results to the FDA (Hitchens, 1995) and USDA–FSIS (Cottingham, 2002) culture-based methods of detection. Method agreement with the TLVIA assay was 94.7% (Knight et al., 1996), while agreement for the Assurance assay was 81% (Feldstine et al., 1997a). Both assays were based on polyclonal antibodies and both are Listeria sp. specific rather than L. monocytogenes specific. All samples were enriched on either LEB (Feldstine et al., 1997a; Knight et al., 1996) or Fraser (Feldstine et al., 1997a) broth prior to analysis by the sandwich ELISA technique. The biggest difference between the two assays is that in the TLVIA assay, the first antibody is bound to the plate before the kit is sold (Knight et al., 1996). A sandwich ELISA is also used in the bListeria-TekQ (Organon Teknika, MD) method described by Durham et al. (1990) and Mattingly et al. (1988). Fifteen monoclonal antibodies were raised to a Listeria-specific antigen. The monoclonal antibodies do not cross react with other bacteria, such as Streptococcus pyogenes, Lactobacillus casei, and Citrobacter freundii, but the antibodies are only genus specific (Mattingly et al., 1988). Mattingly et al. (1988) demonstrated that the Listeria-Tek procedure could detect Listeria sp. from pure culture and from contaminated foods. Durham et al. (1990) demonstrated that the detection of Listeria contamination by the method in different food samples is comparable to the FDA culturing method. An interesting variation on the sandwich ELISA is that proposed by Chemboro et al. (2005). In this paper, antibodies specific for L. monocytogenes were immobilized onto activated charcoal. L. monocytogenes cells in solution were captured and detected by adding HRPconjugated L. monocytogenes-specific antibodies. The flow of peroxidase substrates could be measured as an amperometric signal (method described in (AbdelHamid et al., 1999)). The limit of detection achieved by the authors in pure culture was 10 cfu/ml in 30 min. This assay is specific, but is only amenable as of yet to liquid samples (Chemboro et al., 2005) and is still prohibitively expensive. A competitive ELISA can also be used to detect the presence of pathogens since this method is less

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susceptible to non-specific background binding than the direct or sandwich ELISAs. In this format, a known amount of antigen is coated onto the plate. The sample to be tested is incubated with the primary antibody and then added to the blocked well. After washing, the secondary antibody is added to the well, and detection is performed as mentioned above. In this case, the more free antigen present in the test solution, the less signal will be detected after treatment with the secondary antibody since the majority of the primary antibody will have bound to the soluble antigen, and is consequently washed away. The amount of inhibition can be compared to an inhibition curve created with known amounts of antigen in the samples, thus allowing a quantitative determination of the amount of pathogen present in the test sample via interpolation using the standard curve (Hess et al., 1998). The competitive ELISA format has not been used in the detection of L. monocytogenes. However, in a slight variation, a competitive immunoassay using surface plasmon resonance (SPR) to detect L. monocytogenes has been performed. SPR measures the change in the refraction of a light beam as the mass of a molecule changes when bound by an antibody to detect binding. The procedure has been reviewed in other articles (Bamdad, 1997; Hashimoto, 2000; Markey, 2000). In this case, Leonard et al. (2005) immobilized recombinant InlB protein on a sensor chip. They then incubated anti-InlB polyclonal antibody with varying concentrations of L. monocytogenes and passed the remaining antibodies over the chip. The larger the number of L. monocytogenes cells in the sample, the fewer the antibodies were available to bind to the immobilized InlB. The limit of detection of this method was about 105 cfu/ml. The same authors used a similar technique previously (Leonard et al., 2004). In that case, they preincubated L. monocytogenes with anti-L. monocytogenes antibodies, centrifuged out the bound antibodies, and passed the unbound antibodies over immobilized anti-Fab antibodies. The larger the number of L. monocytogenes cells in the sample, the fewer the anti-L. monocytogenes antibodies were available to bind the coated chip. The limit of detection was the same as the newer paper, but the centrifugation step is time consuming and impractical. The competitive immunoassay-based SPR is fast, but prohibitively expensive and still requires work to increase the sensitivity. 2.6.3. ELFA — enzyme-linked fluorescent assay The ELISA assay can be made more sensitive by conjugating fluorescent labels to the antibodies. This

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type of labelling also decreases the time required for detection by eliminating the need for a colorimetric reaction as the final step of the ELISA. The labelling does, however, increase the cost of the assay. Sewell et al. (2003) used this method to detect L. monocytogenes in a commercial system, VIDAS (bioMe´rieux Vitek), that contains a monoclonal antibody conjugated to a fluorescent reporter dye. The authors compared detection of naturally contaminated food between the culture-based PALCAM method, and the ELFA based VIDAS method. In both methods, various food samples were homogenized and incubated in enrichment culture. In the PALCAM method, the broth was streaked onto modified Oxford or PALCAM agar and incubated for a further 48 h. In the VIDAS method, the enriched culture was heat killed and added directly to the ELFA. Culture enrichment was required to reach the 104–105 cfu/ml needed for detection by the ELFA method. The accuracy of the positive results was 97% with false negative and false positive rates of 1.9% and 3.0%, respectively. Detection time for preliminary positive results was three days rather than five (Sewell et al., 2003). These results have been confirmed by two independent groups (Gangar et al., 2000; Silbernagel et al., 2005). Gangar et al. (2000) showed in a collaborative study that the results from the VIDAS LIS method and the USDA culture-based method were 86% in agreement, while a collaborative study by Silbernagel et al. (2005) demonstrated that the VIDAS LIS method was equivalent to culture based-methods at a 95% confidence interval when the FDA method (Hitchens, 1995) was used for fish and green beans, the USDA method (Cottingham, 2002) was used for cooked roast beef and the AOAC Official Method 993.12 (Anonymous, 2000) was used for ice cream and brie cheese. Dunbar et al. (2003) used a variation of the fluorescent immunoassay for detection of foodborne pathogens. The researchers used microspheres with different spectral labels, coated with antibodies to each of E. coli O157:H7, Salmonella typhimurium, C. jejuni, and L. monocytogenes to detect the pathogens after enrichment (a different spectral label for each type of antibody). The spheres were sorted by their spectral labels using a fluorescent bead sorter (Luminex Labmap system, Austin TX) and the pathogens detected using a secondary antibody labelled with a fluorophore. The assay is able to detect between 2.5 and 500 organisms/ ml depending on the species (Dunbar et al., 2003). This system, although expensive, allows multiple pathogens to be detected simultaneously.

2.6.4. Immunoprecipitation and agglutination assays One simple assay that has been developed for the detection of foodborne pathogens is the latex agglutination assay, which makes use of latex bead-bound antibodies specific to the antigen of choice. Antibodies on each bead bind to the antigen. Each antigen can bind to more than one antibody bead, causing the beads to agglutinate. Matar et al. (1997) used antibodies specific to the L. monocytogenes LLO toxin to detect the presence of this toxin in foods and pure Listeria cultures. The toxin was used instead of cells because antibodies to cell surface proteins tend to be more genus-specific. Additionally, LLO is a secreted protein, so that the supernatants of enrichment cultures can be used directly without further extraction. After culture enrichment following the USDA method, the latex agglutination assay was able to detect LLO at concentrations that indicate contamination of the original food sample with between 0.3 and 220 cfu/g food sample (Matar et al., 1997). The agglutination test itself gives qualitative (i.e., positive versus negative) results (Matar et al., 1997) rather than quantitative results. Although the latex agglutination test still requires culture enrichment and then 48 h to give results, it does have one major advantage over most of the molecular methods available in that no specialized equipment is required. Only visual assessment is required to see the agglutination result. Furthermore, there is no need for any expensive reagents, making the test suitable for use in the field (Matar et al., 1997). Feldstine et al. (1997b) developed an immunoprecipitation method that made use of heat killed L. monocytogenes cells to detect contaminated samples. The authors inoculated various food samples with between 0.003 and 11 cfu/ml, and then performed enrichment culturing. Samples of the secondary enrichment culture were heated to ensure that all L. monocytogenes were dead, and the sample added to the Visual Immunoprecipitate (VIP) device. The theory of this device is that as the enrichment broth flows laterally along the solid support, any Listeria sp. will interact with an antibody–chromogen complex. The complex flows across a lateral flow membrane and is captured by antibody immobilized on the membrane. If Listeria sp. is present in the sample, a coloured line will form across a viewing window. The authors claimed that detection of L. monocytogenes was equivalent to that of the Bacteriological Analytical Manual and the USDA method, but did not separate the detection of L. monocytogenes from other Listeria sp. and in fact used the two terms interchangeably.

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3. Future perspectives L. monocytogenes is an important emerging foodborne pathogen. Although incidences of infection are low, it accounts for approximately 28% of all foodrelated deaths in the United States (Donnelly, 2001; Mead et al., 1999). As a result of this, and because the number of bacteria required to cause infection is unknown, the ability to detect the organism in low numbers in food is essential. For the food industry, the ideal pathogen detection test should have at least some of the following characteristics: simple to perform by non-specialized personnel, sensitive enough to detect low numbers of pathogens, specific for detection of pathogenic species of interest, rapid, amenable to automation, and inexpensive (Ingianni et al., 2001), being able to detect the presence of only live/virulent organisms. Technologies are becoming available that satisfy a number of these criteria, but none as yet has been able to satisfy all. Efforts should be specifically directed to cutting the detection time and increasing the sensitivity and specificity of detection of either the organism or the toxin. One of the best ways to ensure that only the pathogenic L. monocytogenes is detected, rather than the other non-pathogenic species of Listeria, is to detect a virulence factor such as a toxin. In L. monocytogenes, the obvious choice of toxin for detection is LLO. Because the hlyA gene encoding the toxin is only present in the pathogenic species, it is appropriate for detection of L. monocytogenes using nucleic acid-based methods. Also, since the toxin is secreted, it is a good choice for antibody-based detection methods, although culture conditions that ensure the expression of the toxin must be met. With the availability of high yield, easily purified recombinant LLO to serve as the antigen for the production of antibodies (Churchill et al., 2005; Giammarini et al., 2003) and for use in competitive ELISA formats, antibody-based detection methods for the LLO toxin are becoming practical. Although the technologies being developed to detect L. monocytogenes promise to improve the efficiency, sensitivity and specificity of pathogen detection, one cannot overstate the importance of preventing foodborne pathogens from entering the food chain in the first place. Innovations in food production such as hazard analysis critical control point (HACCP) programs play an important role in preventing the spread of the pathogens, as do the efforts of inspectors and consumers in reporting unsafe food handling practices.

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