An 8-hour system for Salmonella detection with immunomagnetic separation and homogeneous time-resolved fluorescence PCR

International Journal of Food Microbiology 125 (2008) 158–161

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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i j f o o d m i c r o

An 8-hour system for Salmonella detection with immunomagnetic separation and homogeneous time-resolved fluorescence PCR Virve Hagren a,⁎, Piia von Lode a, Anniina Syrjälä a, Teemu Korpimäki b, Mika Tuomola b, Otto Kauko a, Jussi Nurmi a a b

Abacus Diagnostica Ltd., Tykistökatu 4D, FI-20520 Turku, Finland Raisio Diagnostics, Joukahaisenkatu 1, FI-20520 Turku, Finland

A R T I C L E

I N F O

Article history: Received 28 December 2007 Received in revised form 12 March 2008 Accepted 28 March 2008 Keywords: Salmonella Immunomagnetic separation Homogeneous assay PCR Time-resolved fluorometry Dry chemistry

A B S T R A C T We describe a system consisting of rapid sample enrichment and homogeneous end-point PCR analysis that enables the detection of Salmonella in various food matrices in 8 h. Sample preparation starts with 6 h enrichment step in supplemented broth, after which Salmonella cells are collected with immunomagnetic particles. The particles are washed and dispensed to ready-to-use PCR reaction vessels, which contain dried assay-specific reagents and an internal amplification control. PCR is performed with a novel instrument platform utilising the sensitive label technology of time-resolved fluorometry. Qualitative assay results are automatically interpreted and available in 45 min after sample addition. The overall accuracy, sensitivity and specificity of the Magda™ CA Salmonella system were 99.1%, 98.4% and 100.0%, respectively, based on the evaluation of 107 samples (beef, pork, poultry and ready-to-eat meals) artificially contaminated with sublethally injured Salmonella cells. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Salmonella is a common foodborne pathogen in humans and animals, and one of the leading causes of foodborne illness (Tirado and Schmidt, 2001). It has been estimated that in the United States alone there are 1.4 million Salmonella infections per year (Voetsch et al., 2004). Typically, infections are transmitted through raw or undercooked foods, such as meat, poultry, eggs and dairy products, or foods containing raw ingredients. In order to reduce the risk of infection, the food production chain is monitored and controlled for various microbiological risks. The control ofSalmonella in theEuropean Union is defined in Regulation 2160/ 2003(Anonymous, 2003a).A recentstudyon theprevalence ofSalmonella in broiler flocks in Europe revealed that on average more than 20% of the flocks were Salmonella positive with a range from 0% to nearly 70% depending on the country (European Food Safety Authority, 2007). In Finland, the effective implementation of the National Salmonella Control Programme(Anonymous,1994) has usually kept theannualoccurrenceof Salmonella in production animals and foodstuffs originating from these at a level of less than 1% (Huttunen et al., 2006). However, in order to maintain this favourable situation, continuous surveillance is required. ⁎ Corresponding author. Tel.: +358 20 7188 385; fax: +358 20 7188 381. E-mail address: [email protected] (V. Hagren). 0168-1605/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2008.03.037

A widely accepted standard for detecting Salmonella in foods is a traditional microbiological method based on a non-selective preenrichment, selective enrichment, subsequent plating on selective media and biochemical identification (Anonymous, 2002). However, culture-based methods are typically time-consuming and it may take several days before the results are obtained. Nucleic acid techniques offer faster detection systems for foodborne pathogens and consequently, a number of nucleic acid-based assays for Salmonella have been developed (for a review, see Mozola, 2006). Although nucleic acid methods are increasingly being utilised as tools in food diagnostics, the implementation of polymerase chain reaction (PCR) in routine use has suffered from extensive manual workload requiring a high level of expertise in molecular biology, expensive instrumentation and lack of robust PCR methods, which can result in inconsistent results between laboratories. Recently, systematic approaches to validation and standardization of PCR protocols have been reported in order to facilitate the application of alternative methods in foodborne pathogen detection (Abdulmawjood et al., 2004; D'Agostino et al., 2004; Malorny et al., 2003a,b,c). The aim of the Magda CA Salmonella system was to address two critical issues in Salmonella detection: to decrease the total analysis time, and in particular, the sample enrichment time and to simplify the DNA-based detection. This study evaluates the performance

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characteristics of the system using sample matrices spiked with sublethally injured Salmonella cells. 2. Materials and methods 2.1. Reagents and instrumentation Magda™ CA Salmonella system, containing elution and wash buffer, enrichment supplement, antibody-coated magnetic particles and PCR kits, was obtained from Raisio Diagnostics (Turku, Finland). GenomEra™ PCR instrument was from Abacus Diagnostica (Turku, Finland). Orbital shaker Heidolph Unimax 1010 equipped with a heating module and a large hood was obtained from Heidolph Instruments (Schwabach, Germany). The shaker was modified with a Magda™ shaker tray for the loading of stomacher bags in Magda™ bag holders (Raisio Diagnostics). PickPen® 8-M was purchased from BioNobile (Turku, Finland). Rappaport Vassiliadis broth (RVS) and xylose lysine desoxycholate (XLD) agar were from Lab M (Bury, UK), buffered peptone water (BPW) from Merck (Darmstadt, Germany), M-broth from BD (Sparks, MD, USA) and tryptic soy agar (TSA) and yeast extract from Sigma-Aldrich (St. Louis, MO, USA). 2.2. Sample matrices The selected sample matrices were raw meat (sliced or minced beef, pork and poultry, n = 74) and ready-to-eat meals (sandwiches, meatballs and chicken pasta, n = 33). The samples were purchased from local supermarkets and stored at 2–8 °C before use. 2.3. Bacterial strains Salmonella and non-Salmonella strains used in selectivity testing are listed in Table 1. The bacteria were pre-cultured aerobically in M-broth (1 colony in 5 ml) overnight at 37 °C. For the inclusivity study, the precultured Salmonella cells were serially diluted in BPW, added to 10 ml of BPW supplemented with Magda CA enrichment supplement to obtain a level of approximately 2000 colony forming units (cfu)/ml and subjected to immunomagnetic separation (IMS) before PCR analysis. The exclusivity of the method was evaluated with non-Salmonella strains plated on TSA, from which single colonies were picked and suspended to water for PCR. The detection limit of the assay was determined with four Salmonella serovars, Typhimurium, Enteritidis, London and Infantis, serially diluted (10-fold dilutions) in BPW. The dilutions were plated on TSA with 6 g/l yeast extract to determine the number of cells and analysed with PCR after subjecting the cells to IMS in 10 ml of supplemented BPW in duplicate. For the performance study, the pre-cultured cells were stressed by heating for 30 min at 55 °C and by freezing for 24 h at −20 °C. Subsequently, the cells were thawed at room temperature and diluted with BPW to appropriate concentration to obtain a spiking level of approximately 1–10 cfu/25 g. The level of sub-lethal injury was determined by plating the cells on two replica plates of TSA with 6 g/l yeast extract and TSA with 6 g/l yeast extract supplemented with 2.5% NaCl and calculated as follows: log (colonies on TSA with 6 g/l yeast extract) − log (colonies on TSA with 6 g/l yeast extract and 2.5% NaCl). The average logarithmic level of sub-lethal injury was 0.34 (SD= ±0.25). 2.4. Sample preparation Each sample was thoroughly mixed prior to division into 25 g portions for PCR analysis and plating. For PCR, the sample was artificially contaminated with the sub-lethally injured Salmonella cells and homogenised in 225 ml of BPW with 1.25 ml of Magda CA enrichment supplement. The sample was incubated in orbital shaker for 6 h at 37 °C, after which a 10 ml sub-sample was collected for IMS. Immunomagnetic particles (10 µl) were added to the sub-sample and incubated for 50 min at 37 °C with shaking. The particles were

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collected with PickPen by inserting the magnetic tip into the subsample for a 10 min period at 37 °C with shaking. The particles were washed twice with 800 µl of wash buffer and once with 200 µl of elution buffer. Finally, the particles were suspended to 60 µl of elution buffer and an aliquot of 30 µl was utilised for PCR. For the culture method, the sample was artificially contaminated with the sublethally injured Salmonella cells and homogenised in 225 ml of BPW.

Table 1 Salmonella and non-Salmonella used in the selectivity study Salmonella serovar (subspecies)

Number of strains

Agona (I) Brandenburg (I) Bredeney (I) Derby (I) Gloucester (I) Heidelberg (I) Indiana (I) Saintpaul (I) Tripoli (I) Typhimurium (I) Blockley (I) Goldcoast (I) Hadar (I) Infantis (I) Kentucky (I) Kottbus (I) Lille (I) Livingstone (I) Mbandaka (I) Montevideo (I) Newport (I) Oranienburg (I) Tennessee (I) Thompson (I) Virchow (I) Dublin (I) Enteriditis (I) Amsterdam (I) Anatum (I) London (I) Senftenberg (I) Abaetetuba (I) Senegal (I) Havana (I) Idikan (I) Kedougou (I) Poona (I) Caracas (I) Cerro (I) Minnesota (I) Nima (I) Urbana (I) Adelaide (I) Wandsworth (I) Johannesburg (I) Tomegbe (I) Koketime (I) 48:z4, z23:- (IIIa) Total

2 2 1 2 1 2 1 2 1 2 1 1 2 1 1 1 1 1 2 2 2 1 1 1 1 1 3 1 2 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 62

Non-Salmonella Citrobacter freundii Enterobacter nimipressuralis Escherichia coli Escherichia fergusonii Hafnia alvei Klebsiella pneumoniae Listeria innocua Listeria ivanovii Listeria monocytogenes Proteus mirabilis Proteus vulgaris Total

1 1 1 1 1 1 1 1 1 1 2 12

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The sample was incubated for 16 h at 37 °C, after which an aliquot of 0.1 ml was collected and added to 10 ml of RVS. After 24 h incubation at 41.5 °C, 0.1 ml was plated on XLD and the plate was incubated for 24 h at 37 °C. The serological confirmation was performed with polyvalent Salmonella antisera according to the manufacturer's instructions (Denka Seiken, Tokyo, Japan).

tion (Anonymous, 2003b). No false positive or inconclusive results were obtained. One false negative result in the analysis of ready-to-eat meals resulted in an overall accuracy of 99.1%. The result was not caused by PCR inhibition, because the internal amplification control (IAC) was amplified. However, the false negative might be due to the low spiking level using sub-lethally injured cells, which could have affected the efficiency of the enrichment.

2.5. PCR assay 4. Discussion The assay was performed with a novel PCR instrument, GenomEra (Hagren et al., 2008). The assay protocol, described in detail elsewhere (Hagren et al., 2008), had a total turn-around-time of 45 min. In brief, all the assay-specific reagents were pre-dispensed and dried in the reaction vessel, which was labelled with a barcode containing the information about the assay protocol and vessel type (control or sample). The assay was simply started by adding the immunomagnetic particles used in the sample preparation to the vessel and inserting the vessel to the instrument. During thermal cycling the irreversibly sealed vessel was transferred between thermal blocks set at constant temperatures. The homogeneous, closed tube end-point detection was based on the use of lanthanide chelate labelled probes and time-resolved fluorometry (Nurmi et al., 2007). The dedicated software of the instrument automatically interpreted the qualitative assay result – positive, negative or inconclusive – based on predetermined lot-specific cut-off values. 3. Results The selectivity of the IMS and PCR was evaluated with 62 Salmonella strains and 12 non-Salmonella strains (Table 1). Inclusivity is defined as the ability of the method to detect the target analyte from a wide range of strains and exclusivity as the lack of interference from non-target strains (Anonymous, 2003b). All the analysed Salmonella strains gave positive results and non-Salmonella negative results in PCR, thus both inclusivity and exclusivity of the assay were 100%. The detection limit, evaluated with four commonly occurring Salmonella serovars, was less than 2.5 cfu/ml. This was based on the determination of the required cell density at the end of the enrichment step, which could still produce a positive result in PCR. Accordingly, the sensitivity of the assay was affected by both IMS and PCR. The performance of Magda CA Salmonella system was compared to a slightly modified culture-based method (Anonymous, 2002). A total of 107 food samples were analysed (Table 2). Negative samples (n = 44) were prepared in parallel with samples (n = 63), which were artificially contaminated with various strains of sub-lethally injured Salmonella. Because the acquisition of naturally contaminated samples is difficult in Finland, artificial contamination with sub-lethally injured cells was used to mimic the situation in naturally contaminated food samples. Accuracy, sensitivity and specificity were calculated from the results according to guidelines by International Organization for Standardiza-

Table 2 Results of the comparison study Sample type

PA

NA

PD

ND

N

AC%

N+

SE%

N−

SP%

Meat Ready-to-eat meals

44 18

30 14

0 0

0 1

74 33

100.0 97.0

44 19

100.0 94.7

30 14

100.0 100.0

N = total number of samples [PA + NA + PD + ND]. N+ = total number of positive results with the reference method [PA + ND]. N− = total number of negative results with the reference method [NA + PD]. PD = positive deviation (number of samples that are PCR positive and reference negative). ND = negative deviation (number of samples that are PCR negative and reference positive). PA = positive agreement (number of samples that are PCR positive and reference positive). NA = negative agreement (number of samples that are PCR negative and reference negative). AC% = relative accuracy [(PA + NA) / N × 100]. SE% = relative sensitivity [(PA / N+) × 100]. SP% = relative specificity [(NA / N−) × 100].

IMS, based on the use of paramagnetic particles or beads coated with antibodies against the surface antigens of the target cell, can be used to concentrate and separate the target organisms from the background flora (for a review, see Olsvik et al., 1994; Safarik and Safarikova, 1999). However, traditional IMS can be rather laborious and therefore, it is not ideal for processing large number of samples. Nevertheless, the use of PickPen device in the magnetic separation has been reported to accelerate sample preparation, to reduce carryover of background flora and to enhance recovery of the particles (Nou et al., 2006). Previously, IMS of Salmonella from various food matrices has been combined with identification systems, such as PCR (Chen and Griffiths, 2001; Fluit et al., 1993; Hsih and Tsen, 2001; Notzon et al., 2006; Widjojoatmodjo et al., 1992), immunoassays (Cudjoe et al.,1995; Gehring et al.,1996; Holt et al., 1995) and plating (Cudjoe et al., 1994; Mansfield and Forsythe, 1996; Ripabelli et al., 1997). In the current system, Salmonella cells were specifically collected from a 10 ml sample with PickPen and immunomagnetic particles after 6 h incubation. The sample volume utilised in the collection of the cells affected the sensitivity of the assay. Typically, a larger sample volume contains more target cells, and thus, provides a more representative sample. In addition to the efficient IMS, the enrichment phase was modified to provide optimal growth conditions for Salmonella. This was achieved in particular by supplementing the growth medium and by optimising the aeration through shaking. PCR was carried out using a novel PCR instrument platform, GenomEra, which contained a PC-controlled thermocycler with timeresolved fluorometer. To our knowledge, this is the first PCR instrument capable of implementing homogeneous time-resolved fluorescence nucleic acid assays. The homogeneous assay concept is faster, less laborintensive and less prone to contamination than the conventional separation-based assays (Foy and Parkes, 2001). Lanthanide chelates in combination with temporal separation in the measurement phase provide basis for detection technology known as time-resolved fluorometry (Soini and Lövgren, 1987), which can bring along a 100fold improvement in assay sensitivity compared to conventional label technologies (Nurmi et al., 2002). In particular, the use of intrinsically fluorescent lanthanide chelates enables homogeneous detection, where the signal is measured directly from the liquid phase without any separation steps (von Lode et al., 2003). In general, ready-to-use mixtures in PCR facilitate automation, reduce consumption of materials and offer simplicity, speed and reproducibility to the assay. Moreover, risks of human error and PCR contamination are significantly reduced. In order to simplify the assay procedures and instrumentation, ready-to-use reaction vessels containing all the assayspecific reagents in dry form were utilised in the current assay. The stability of these dried mixtures was shown to be at least 6 months at −20 °C or 4 months at room temperature (Hagren et al., 2008). The assay procedure was simple, since the user only had to add the sample to the vessel and transfer the vessels to GenomEra instrument. The selection of the correct assay protocol was done automatically by the software based on the barcodes on the vessels. Prior to thermal cycling, the instrument heat-sealed the vessels irreversibly, which minimised the risk of carryover contamination. After cycling, the software deduced the qualitative end results based on the time-resolved fluorescence measurements and lot-specific cut-off values. Sample was classified as positive, if the calculated result was higher than the cut-off and vice versa. An inconclusive result was obtained, if neither Salmonella nor IAC,

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which was included in every reaction, was amplified. Generally, the inclusion of IAC in PCR is advisable for two reasons; IAC can reveal a false negative result due to PCR failure and it can be used for quantitation, if required (Hoorfar et al., 2004; Zimmermann and Mannhalter, 1996). In food analysis, particularly fat and proteins are associated with PCR inhibitory effects (Rossen et al., 1992; Wilson, 1997). In principle, IMS can reduce PCR inhibition, because the antibody-coated particles should bind only the target cells and during the washing protocol any remaining nonspecifically bound material should be removed. Typically, traditional analysis methods are slow and/or expensive and can create delays in the food production chain. The main advantage of the described system is the speed, since the results from the entire process, starting from the sample enrichment and ending to the PCR analysis, can be obtained within one work shift. This is a clear advantage for the food industry in many ways. For example, storage times can be reduced and in case of positive results, cleaning activities can immediately be started and contaminated batches removed from the food chain. This screening method can easily be implemented in laboratories with no previous expertise in nucleic acid assays, because the simplified homogeneous dry chemistry assay concept minimises the amount of manual steps in PCR into only one pipetting step. Altogether, Magda CA Salmonella system is applicable to routine use and it also promotes testing in low-volume settings, such as on-site food monitoring. Acknowledgement This study was partially financed by the Finnish Funding Agency for Technology and Innovation (TEKES). References Abdulmawjood, A., Bulte, M., Roth, S., Schonenbrucher, H., Cook, N., D'Agostino, M., Malorny, B., Jordan, M., Pelkonen, S., Hoorfar, J., 2004. Toward an international standard for PCR-based detection of foodborne Escherichia coli O157: validation of the PCR-based method in a multicenter interlaboratory trial. Journal of AOAC International 87, 856–860. Anonymous, 1994. 94/968/EC: Commission Decision of 28 December 1994 approving the operational programme for the control of Salmonella in certain live animals and animal products presented by Finland. Official Journal L371, 36–37. Anonymous, 2002. Microbiology of food and animal feeding stuffs — Horizontal method for the detection of Salmonella spp. (ISO 6579:2002). International Organization for Standardization, Geneva, Switzerland. Anonymous, 2003a. Regulation (EC) No 2160/2003 of the European Parliament and of the Council of 17 November 2003 on the control of salmonella and other specified food-borne zoonotic agents. Official Journal L325, 1–15. Anonymous, 2003b. Microbiology of food and animal feeding stuffs — Protocol for the validation of alternative methods (ISO 16140:2003). International Organization for Standardization, Geneva, Switzerland. Chen, J., Griffiths, M.W., 2001. Detection of Salmonella and simultaneous detection of Salmonella and Shiga-like toxin-producing Escherichia coli using the magnetic capture hybridization polymerase chain reaction. Letters in Applied Microbiology 32, 7–11. Cudjoe, K.S., Krona, R., Olsen, E., 1994. IMS — a new selective enrichment technique for detection of Salmonella in foods. International Journal of Food Microbiology 23, 159–165. Cudjoe, K.S., Hagtvedt, T., Dainty, R., 1995. Immunomagnetic separation of Salmonella from foods and their detection using immunomagnetic particle (IMP)–ELISA. International Journal of Food Microbiology 27, 11–25. D'Agostino, M., Wagner, M., Vazquez-Boland, J.A., Kuchta, T., Karpiskova, R., Hoorfar, J., Novella, S., Scortti, M., Ellison, J., Murray, A., Fernandes, I., Kuhn, M., Pazlarova, J., Heuvelink, A., Cook, N., 2004. A validated PCR-based method to detect Listeria monocytogenes using raw milk as a food model — Towards an international standard. Journal of Food Protection 67, 1646–1655. European Food Safety Authority, 2007. Report of the Task Force on Zoonoses Data Collection on the Analysis of the baseline survey on the prevalence of Salmonella in broiler flocks of Gallus gallus, Part A. The EFSA Journal 98, 1–85. Fluit, A.C., Widjojoatmodjo, M.N., Box, A.T.A., Torensma, R., Verhoef, J., 1993. Rapid detection of salmonellae in poultry with the magnetic immuno-polymerase chain-reaction assay. Applied and Environmental Microbiology 59, 1342–1346.

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