Transia Plate Salmonella Gold for the detection of Salmonella spp. in foods and feeds

Transcription

1 Transia Plate Salmonella Gold for the detection of Salmonella spp. in foods and feeds Abstract A method for the detection of Salmonella spp. in foods and feeds is presented. This method, which is based on a two step enrichment followed by a sandwich-type immunoassay, produces a presumptive positive result within 36 to 40 hours of analysis. The inclusivity and exclusivity of the test were assessed on 129 Salmonella strains from 101 different serovars and 72 strains from 50 non-salmonella species; these results demonstrated the high levels of inclusivity (96.1 %) and exclusivity (100 %) of the antibodies used in the assay. The overall method agreement calculated on confirmed results between Transia Plate Salmonella Gold (TPSG) and the ISO 6579:2002 methods was 98.8 %. With a sensitivity rate of 99.3 % and a specificity rate of 97.4 % the Transia Plate Salmonella Gold method appeared to give comparable results to the reference method. The high specificity of the antibodies used in the method was confirmed by the absence of false positive reactions, as all the positive results obtained with the Transia test were confirmed by streaking the RVS enrichment broth onto selective agars. The ruggedness of this method was studied in this work and showed that the length of the heat inactivation step, the reagents volumes and incubation parameters could be exceptionally modified without any significant effect on the results. Method Authors Patrice Arbault, V. Buecher, S. Poumerol and M.-L. Sorin Diffchamb S.A., 8 rue Saint-Jean de Dieu, Lyon, France Submitting Company Diffchamb AB, F O Petersons Gata 32, SE Västra Frölunda, Sweden Independent Laboratory Campden & Chorleywood Food Research Association, Chipping Campden, Gloucestershire, GL55 6LD, United Kingdom Reviewers Michael Brodsky (Brodsky Consultants, Canada) Joseph Odumeru (University of Guelph, Canada) Thomas Hammack (FDA CFSAN, USA) 1

2 1. Scope of Method 1.1 Target organisms Salmonella spp. 1.2 Tested matrices Cooked chicken, raw milk, cantaloupe, sausages, raw shrimps, yogurt, mayonnaise, shell eggs, frozen red berries & currants, bean sprouts, raw ground beef, smoked fish (trout), fresh pasta, milk chocolate, ground black pepper, cake mix, dry milk-based infant formula, dry food for cat, raw ground turkey and Brie cheese. 1.3 Summary of validated performance claims The Transia Plate Salmonella Gold assay for detection of Salmonella spp. in foods and feeds has been tested internally and externally and has shown 99.3 % sensitivity and 97.4 % specificity. The external study demonstrated that the Transia Plate Salmonella Gold method is equivalent to the ISO 6579:2002 reference method. The use of Transia Plate Salmonella Gold assay allows the user to get a presumptive result of analysis within a total time of 36 to 46 hours, which is a significant improvement compared with the time needed by the reference method (minimum one day more) to give the same kind of information. Indeed, quicker reliable results allow food business operators to release fresh products from production one day earlier in case of negative result. In case of positive result these operators can react earlier and thus limit the possible outcomes of the contamination. The excellent specificity of the antibodies used in the assay minimises the risk of false positive reactions and therefore improves the reliability of the screening by reducing the risks of inopportune and non-relevant production chains shutdown or product recalls. 2. Definitions 2.1 Inclusivity: Inclusivity rate = 100 x (No. of pure Salmonella cultures positive with Transia Plate) / (Total No. of pure Salmonella cultures tested). 2.2 Exclusivity: Exclusivity rate = 100 x (No. of pure non-salmonella cultures negative with Transia Plate) / (Total No. of pure non-salmonella cultures tested). 3. Principle Transia Plate Salmonella Gold is a sandwich-type immunoassay relying on a polyclonal antibody coated onto the solid phase (wells of a microtitre plate with divisible strips) and a monoclonal antibody labeled with a peroxydase. The monoclonal antibody recognizes specifically a lipopolysaccharide determinant. The sample (enriched broth) is added to the well after a heat inactivation step to release the Salmonella antigens from the culture possibly present in the sample. After a three (3) step protocol whose total incubation time is 105 minutes, the reading of the microtitre plate is performed with a spectrophotometer at 450 nm. 2

3 4. General Information Salmonella is the leading foodborne bacteria for human enteritis worldwide through the consumption of a variety of contaminated foods, such as eggs, meats and seafood. As Salmonella is isolated from many different matrices, a very large number of food producers are looking for the bacteria in their raw material, manufacturing lines and in their finished products. Today, Salmonella accounts for the largest number of foodborne pathogen analysis worldwide and represents a significant workload. The detection methods for Salmonella are numerous and rely on different technologies, from the cultural methods to the immunoassays and genomic tests. However, some of these methods have not been adequately developed for the routine screening of many food samples. The drawbacks are many and method related. For example, the cultural methods are time-consuming and require a lot of plating with subjective result interpretation. On the other hand PCR-based assays offer a quicker answer, but require a tedious sample preparation followed by cumbersome amplification and detection steps. Immunoassays have been developed for years but some require long enrichment protocols or are known to generate false positive answers due to a lack of specificity. 5. Test Kit Information 5.1 Kit Name: Transia Plate Salmonella Gold. The kit includes 1 microtitre plate (96 wells in 12 divisible 8 wells strips) or 10 microtitre plates (960 wells in 12 divisible 8 wells strips). 5.2 Catalog Number: SA0180 (96 wells), SA0190 (960 wells) 5.3 Ordering Information: From the U.S.: Diffchamb Inc. c/o RAISIO STAEST US, INC. 325 Deming Way Summerville, SC USA Phone (ext. 7103) or DIFFCHAMB Fax sales@diffchamb.com Website address: From abroad: Diffchamb AB F O Petersons Gata Västra Frölunda Sweden Phone Fax order@diffchamb.com 5.4 Test Kit Reagents: Negative control ready to use 1 x 6 ml (SA0180), 2 x 15 ml (SA0190) Positive control: LPS (lipopolysaccharide) ready to use 1 x 3 ml (SA0180), 2 x 8 ml (SA0190) Conjugate: anti-salmonella antibodies conjugated to peroxydase ready to use 1 x 15 ml (SA0180), 4 x 30 ml (SA0190) Washing buffer concentrated 20X 1 x 60 ml (SA0180), 1 x 800 ml (SA0190) Substrate: Urea-H 2 O 2 1 x 10 ml (SA0180), 3 x 30 ml (SA0190) Chromogen: TMB (Tetramethylbenzidine) 1 x 10 ml (SA0180), 3 x 30 ml (SA0190). 3

4 5.4.7 Stop solution: H 2 SO 4 ready to use 1 x 15 ml (SA0180), 5 x 30 ml (SA0190). 6. Additional Media and Reagents 6.1 Enrichment: Distilled water Buffered peptone water Rappaport-Vassiliadis Soya broth (RVS). 6.2 Confirmation of positive results: Isolation: Selective agar plate such as xylose lysine deoxycholate (XLD) or brilliant green agar (BGA) Identification: Biochemical identification gallery (such as API 20E, BioMérieux, Art. No or Microbact 24E, Medvet, Art. No. 1074). 7. Equipment 7.1 Scales (1 to 500 g ± 0.1g) and weighing vessels. 7.2 Homogeniser or mixer (Stomacher Seward 400C). 7.3 Filter-fitted stomacher bags. 7.4 Tubes (20 ml) for the subcultures. 7.5 Magnetic stirrer. 7.6 Air Incubator capable of maintaining 37 ± 1 C. 7.7 Air Incubator capable of maintaining 41.5 ± 0.5 C (or preferably a water bath with circulating water). 7.8 Vortex. 7.9 Screw-cap tubes (5 or 10 ml) resistant to +100 C Water bath 100 C (boiling water) Micropipette µl Micropipette µl (optional) Wash bottle (or preferably an automatic microplate washer) Absorbent paper Multipipette with 2.5 and 5 ml tips. 4

5 7.16 Microplate reader single or double beam reading 450 nm filter and reference filter 595 nm Sterile transfer pipettes (1 ml ± 0.05 ml) 8. Standard Reference Material ISO 6579:2002, Microbiology of Food and Animal Feeding Stuffs Horizontal Method for the Detection of Salmonella spp. Feldsine, P., Abeyta, C., & Andrews W.H. (2002) AOAC International Methods Committee Guidelines for Validation of Qualitative and Quantitative Food Microbiological Official Methods of Analysis, J. AOAC Int. 85, Safety Precautions Good laboratory practice should be employed when using the kit. Safety clothing should be worn and skin contact with reagents avoided. Material safety data sheet available on request. Contaminated material should be disposed according to local, state and federal regulations. 10. Sample Preparation 10.1 Homogenise 25 g or 25 ml of the sample with 225 ml of buffered peptone water in a stomacher bag. Use of a filter-fitted stomacher bag is recommended Incubate at 37 ± 1 C for hours Homogenize and inoculate 0.1 ml of the pre-culture broth in 10 ml of RVS Incubate for hours at 41.5 ± 0.5 C Heat 1 to 2 ml of the enrichment broth in a tube in a water bath at 100 C (boiling water) for 20 minutes, then cool to room temperature. Keep the rest of the enrichment broth at 3 ± 2 C should confirmation of a positive result prove necessary later. 11. Analysis The test should be performed at room temperature (18-25 C). Remove the reagents from the box and allow them to come to room temperature at least one hour before use. Have all reagents and samples ready for use so that the various materials can be added to the wells without delay. Shake each sample manually or using a Vortex before use. 5

6 11.1 Attach the required number of strips to the plate: two wells for the negative control, one for the positive control and one well for each sample. Return the unused strips to the zip-lock bag with dehydrating agent and close it tightly. Write the position of the controls and samples on the working sheet Distribute 100 µl of the controls and the samples into the assigned wells. Cover the plate. Controls and samples 11.3 Incubate at room temperature (18-30 C) for one hour. Prepare the washing buffer Holding the plate firmly, shake out the contents of the plate over paper towelling by briskly flicking the wrist. Rinse each well; keep the washing buffer in the wells for one minute (for the first two washings). Empty the plate over a container and then remove the washing buffer by inverting the plate on a paper towel and tapping the plate firmly several times. Repeat the washing five times Add 100 µl of conjugate (vial 4) to each well. Be careful not to touch the wells with the dispensing tip. Cover the plate. Conjugate 11.6 Incubate at room temperature (18-30 C) for 30 minutes. Just before the end of this incubation stage, prepare the required volume of substrate/chromogen mixture using the following formula: required volume = (60 µl substrate + 60 µl chromogen) x number of wells used] Wash the microplate 5 times as indicated in paragraph Add 100 µl of the substrate/chromogen mixture to each well using a multipipette and cover the plate. Discard any unused mixture. The chromogen and substrate can be added without prior mixing - add 50 µl of substrate (vial 5) and 50 µl of chromogen (vial 6) to the wells. Substrate/chromogen mixture 6

7 11.9 Incubate at room temperature (18-30 C) for 15 minutes Add 100 µl of stop solution (vial 7) to each well following the same order used when the substrate/chromogen were added. Mix the contents of the wells thoroughly to ensure complete color conversion. The blue turns to yellow. Stop solution Read the optical densities at 450 nm using a microtiterplate reader (blank on air), use a reference filter 595 nm for double beam reading. 12 Result interpretation 12.1 Validation of the test The optical density of the positive control (PC) must be higher than or equal to The optical density of the negative control (NC) must be lower than or equal to (for double beam reading) or (for single beam reading). If the controls do not meet these requirements, the results are invalid Positive threshold The positive threshold is calculated as the average of the negative controls plus 0.11: [(NC1 + NC2) / 2] Negative threshold The negative threshold is calculated as the positive threshold multiplied by Positive samples A sample is considered positive for Salmonella if its optical density is equal to or higher than the positive threshold 12.5 Negative samples A sample is considered negative for Salmonella if its optical density is lower than the negative threshold 12.6 Doubtful samples A sample is considered doubtful if its optical density is lower than the positive threshold but equal to or higher than the negative threshold 12.7 Confirmation of a doubtful or positive result A positive or doubtful result should be confirmed by streaking the non-heated RVS broth onto selective media plates followed by biochemical identification (see section 6.2, Confirmation of positive results). 7

8 13 Internal Validation Studies Internal validation studies were conducted at the R&D Centre of the Diffchamb Group (Diffchamb S.A. Lyon, France) Inclusivity Testing One hundred twenty nine Salmonella strains from 101 different serovars were analysed to determine the inclusivity of the Transia Plate Salmonella Gold kit (see Table 1) Methodology From a 20-hour culture in BPW (buffered peptone water) at 37 ± 1 o C, each Salmonella strain was subcultured (0.1 ml into 10 ml) in RVS (Rappaport Vassiliadis soya broth) at 41.5 ± 0.5 o C for hours. Following enrichment, 1 ml of each broth was taken and heat-treated in a water bath at 100 o C for 20 minutes. The heat-treated samples were then processed as described in the Transia Plate Salmonella Gold test kit insert. Each unboiled enrichment broth was serially diluted in PBS (Phosphate buffer saline broth) and appropriate dilutions were spread-plated onto TSYEA (Trypticase soya yeast extract agar). The plates were incubated at 37 ± 1 o C for 24 ± 3 h Results Inclusivity results are summarised in Table 3. Among the 129 strains tested, Transia Plate Salmonella Gold correctly detected 124, resulting in an overall inclusivity of 96.1 % Exclusivity Testing Seventy-two (72) strains from 50 different species belonging to non-salmonella genera (Table 2) were analyzed to determine the exclusivity of the kit Methodology From a 20-hour culture in BPW (buffered peptone water) at 37 ± 1 o C, each non-salmonella strain was subcultured (0.1 ml into 10 ml) in BPW at 37 ± 1 o C for hours. Following enrichment, 1 ml of each broth was taken and heat-treated in a water bath at 100 o C for 20 minutes. The heat-treated samples were then processed as described in the Transia Plate Salmonella Gold test kit insert. Each unboiled enrichment broth was serially diluted in PBS (Phosphate buffer saline broth) and appropriate dilutions were spread-plated onto TSYEA (Trypticase soya yeast extract agar). The plates were incubated at 37 ± 1 o C for 24 ± 3 h Results Exclusivity results are summarised in Table 3. All 72 strains tested have been detected as negative with Transia Plate Salmonella Gold, resulting in an overall exclusivity of 100 %. 8

9 13.3 Method Comparison Study Matrix The purpose of this study was to compare the Transia Plate Salmonella Gold method with the ISO 6579:2002 method for the detection of Salmonella spp in eighteen (18) different matrices Methodology Selected strains As agreed with AOAC R.I. the following combinations matrix/strain was tested: Total count (cfu/g) Strain Reference Origin Cooked chicken 2 x 10 4 Salmonella Kentucky 305 AFSSA France Raw milk 5.4 x 10 6 Salmonella Dublin 1236 Dairy Denmark Cantaloupe 3.5 x 10 5 Salmonella Poona 781 Pasteur Institute Paris France Sausages 4.4 x 10 7 Salmonella Bredeney 1656 Pasteur Institute Lille France Raw shrimps 8.1 x 10 8 Salmonella Montevideo 1449 NCTC 5747 Yogurt 1 x 10 2 Salmonella Typhimurium 1654 Pasteur Institute Lille France Mayonnaise < 10 2 Salmonella Heidelberg 1689 Isolated from chicken gizzard France Shell eggs 2.7 x 10 4 Salmonella Enteritidis 1535 Isolated from cocoa - France Frozen red berries & currants 2 x 10 3 Salmonella Newport 1442 Isolated from chicken wings France Bean sprouts 4.5 x 10 8 Salmonella Infantis 1305 Service laboratory Australia Raw ground beef 1.5 x 10 9 Salmonella Anatum 1622 Service laboratory UK Smoked fish (trout) 6.9 x 10 7 Salmonella Virchow 1451 Isolated from chicken filet France Fresh pasta < 10 2 Salmonella IIIb 61:r: Isolated from lamb tongue France Milk chocolate 1.2 x 10 2 Salmonella Senftenberg 1486 Dairy UK Ground black pepper 6.6 x 10 4 Salmonella Indiana 1428 Isolated from cow liver France Cake mix 8.2 x 10 3 Salmonella Thompson 306 Food Inspection Services France Dry milk-based infant formula < 10 2 Salmonella Blockley 1100 Isolated from chicken gizzard France Dry food for cat 7 x 10 2 Salmonella Mbandaka 1618 Service laboratory UK Inoculum preparation Each strain was cultured in M broth (Merck; Darmstadt, Germany), incubated overnight at 37 C. 0.1 ml was subcultured into 5 ml of M broth, for about 4-6 hours at 37 C. This culture was then serially diluted in PBS. Dilutions were used for moist foods contamination. Strains used for the inoculation of processed foods were stressed by heat (10 minutes at 50 C), prior to dilution in PBS. 9

10 For dry foods contamination, a freeze-dried bacterial culture was grinded to a fine powder and added to g of the matrix. After storage at room temperature for 1-2 weeks, the cell density was estimated, using serial dilutions and plating on Tryptone Soya Yeast Extract Agar, by difference between the Total Plate Count of the inoculated and uninoculated matrices. This preparation was used to inoculate two 1000 g portions of each dry food matrix Inoculation procedure Two 1000 g portions of each matrix were inoculated with a dilution of the culture or an aliquot of dry food, estimated to give, for the first one, an inoculum level of 1-10 cfu/25g (3 cfu/25g was aimed) and for the second one, an inoculum level of cfu/25g (30 cfu/25g was aimed), after 3 days of storage. A further 500 g portion of each food remained uninoculated. The inoculated and uninoculated food portions were stored for hours at 2-8 C for moist foods and at room temperature for dry foods, prior to testing. After storage, 20 x 25 g samples from the two inoculated food portions and 5 x 25 g samples from the uninoculated food were weighed out for each matrix. Also, further samples were taken for MPN analysis (3 x 100 g, 3 x 10 g, 3 x 1 g and 3 x 0.1 g). Samples were then tested by the ISO 6579:2002 method for the detection of Salmonella spp and by the Transia Plate Salmonella Gold Test, as briefly described in the next paragraph ISO 6579:2002 method for the detection of Salmonella spp. For the pre-enrichment of all food matrices, samples (including MPN samples) were, after homogenization with a stomacher, enriched in Buffered Peptone Water (BPW, Biokar BM01008) and incubated at 37 C for 18 ± 2h (see Appendix 1). Following pre-enrichment, 1ml of each sample was subcultured into 10ml Muller-Kaufmann Tetrathionate-novobiocin broth (MK, Oxoid CM343) and 0.1ml into 10ml Rappaport Vassiliadis Soya Peptone broth (RVS, Biokar BM07408). RVS broths were incubated at 41.5 C for 21-24h and MKTTn broths were incubated at 37 C for 24 ± 3h. After incubation, a loopful of each selective enrichment broth was streaked onto Xylose Lysine Desoxycholate Agar (XLD, Oxoid CM469) and Brilliant Green Agar (BGA, Biokar BK091HA). Plates were incubated at 37 C for 24h ± 3h. Plates were then examined for typical Salmonella colonies. Upto five (5) typical Salmonella colonies per sample were confirmed by testing the presence of Salmonella O-antigens by slide agglutination using Salmonella O antisera and biochemically using Microbact 24E system (Medvet Australia, article nr 1074). 10

11 The MPN of Salmonella present in the initial samples on the day of analysis was determined by referring to the MPN tables in the Bacteriological Analytical Manual (8 th edition) Transia Plate Salmonella Gold Test The Transia Plate Salmonella Gold Test was performed on the RVS selective enrichment broths used in the ISO method (see Appendix 1). Following 21-24h incubation, 1 to 2ml of each sample was taken and heattreated in a water bath at 100 C for 20 minutes. The heat-treated samples were then processed as described in the Transia Plate Salmonella Gold test kit insert. Samples found to be positive were confirmed according to the ISO method; typical Salmonella colonies observed on XLD and BGA plates streaked from unboiled RVS broths were confirmed by testing the presence of Salmonella O-antigens by slide agglutination using Salmonella O antisera and biochemically using Microbact 24E system (Medvet Australia, article nr 1074) Results All the results are summarized in Table 4 and shown full in Appendix 2 to 19. None of the uninoculated samples were found to contain Salmonella by the two methods. TPSG gave totally equivalent detection results compared to ISO method for all matrices contaminated with low levels of Salmonella except for raw milk. Indeed, for raw milk samples, TPSG gave six positive and two false negative samples, compared to ISO method. ISO method gave five positive and three false negative samples, compared to the Transia method. In total, the two methods gave eight positive samples for raw milk Conclusion The Transia Plate Salmonella Gold test successfully detected low levels of Salmonella in the eighteen (18) food matrices and globally results between the two methods are similar. For all food matrices, except the raw milk, Transia Plate Salmonella Gold test and ISO method gave totally identical results. A few disaccording results were observed with raw milk but are not significant due to the extremely low inoculation level (1 cfu/25 g) Lot-to-lot and Stability Study Lot-to-lot consistency The lot-to-lot consistency was assessed by testing in duplicate three (3) negative controls and three (3) dilutions of two (2) different Salmonella cultures for three (3) different kit lots. One lot was tested at the date of release, the second 40 days after release and the third 9 months after release Methodology Positive test strains: From a 24-hour culture at 37 ± 2º C in BPW (Buffered Peptone Water), each Salmonella strain (No and 1435 from the Diffchamb collection) was subcultured (0.2 ml into 10 ml) in 11

12 RVS (Rappaport Vassiliadis Soya broth) for 24 h ± 2 hours at 41.5 ± 0.5 o C. The RVS was then split in two parts: - one was serially diluted in sterile PBS (Phosphate Buffer Saline) and successively 1 ml of each appropriate dilution and 15 ml of TSYE agar (cooled to 45 ± 1º C) were poured in a Petri dish (3 replicates per dilution). After homogenization and solidification of the agar, the Petri dishes were inverted and incubated for 24 to 48 hours at 37 ± 2º C before enumeration. - the second part was immediately inactivated (20 minutes at 100º C) and refrigerated. After reading of the Petri dishes and estimation of the cell count, the inactivated subculture of Salmonella was adjusted to the requested levels (1x10 5, 5x10 5 and 1x10 6 cells/ml) by dilution in RVS before being processed as described in the Transia Plate Salmonella Gold test kit insert. Negative test strains: From a 24-hour culture in BPW, each non- Salmonella strain (No. 1693, 1694 and 1771 from the Diffchamb collection) was subcultured (0.2 ml into 10 ml) in BPW for 24 h ± 2 hours at 41.5 ± 0.5 o C. The cell count was then estimated by reading the optical density at 470 nm and the cultures were adjusted to 10 9 cells/ml by dilution in BPW before a heat inactivation of 20 minutes at 100 C Results The results from the lot-to-lot consistency study are summarised in Table 5. They show clearly that the three (3) lots analysed gave similar results. The strain nr 1431 (Salmonella Heidelberg) was found negative in all cases at 1x10 5 cells/ml but always positive at the higher levels. The strain nr 1435 (Salmonella Enteritidis) was found slightly positive (just above the positive threshold) in all cases at 1x10 5 cells/ml and always significantly positive at the higher levels. The non-salmonella strains did not generate any signal. In every case the interpretation was the same within the duplicates Stability The stability was assessed by following a kit lot over a 31-day period. Testing was performed on the release date and then 14 and 31 days after, in duplicate with a negative control (non-salmonella cultured broth), and three (3) dilutions of two (2) different Salmonella cultures. This stability study allowed us to compare 2 different storage temperature conditions for kits stored at 37 ± 2 C and 5 ± 3 C for a period of 14 and 31 days. Tests were performed in parallel on the same set of samples Methodology Positive test strains: From a 24-hour culture at 37 ± 2º C in BPW (Buffered Peptone Water), each Salmonella strain (No and 1435 from the Diffchamb collection) was subcultured (0.2 ml into 10 ml) in 12

13 RVS (Rappaport Vassiliadis Soya broth) for 24 h ± 2 hours at 41.5 ± 0.5 o C. The RVS was then split in two parts: - one part was serially diluted in sterile PBS (Phosphate Buffer Saline) and successively 1 ml of each appropriate dilution and 15 ml of TSYE agar (cooled to 45 ± 1º C) were poured in a Petri dish (3 replicates per dilution). After homogenization and solidification of the agar, the Petri dishes were inverted and incubated for 24 to 48 hours at 37 ± 2º C before enumeration. - the second part was immediately inactivated (20 minutes at 100º C) and refrigerated. After reading of the Petri dishes and estimation of the cell count, the inactivated subculture of Salmonella was adjusted to the requested levels by dilution in RVS before being processed as described in the Transia Plate Salmonella Gold kit insert. Negative test strain: From a 24-hour culture in BPW, a Citrobacter diversus strain (No from the Diffchamb collection) was subcultured (0.2 ml into 10 ml) in BPW for 24 h ± 2 hours at 41.5 ± 0.5 o C. The cell count was then estimated by reading the optical density at 470 nm and the cultures were adjusted to 10 9 cells/ml by dilution in RVS before a heat inactivation of 20 minutes at 100 C Results The results of the stability study are summarised in Table 6. They show that the results obtained with all the positive and negative controls used led to the same interpretation even after 31-day storage at 37 ± 2 o C. These conditions, which were selected in order to generate accelerated stability data, did not create significant differences compared with the normal refrigerated storage conditions (5 ± 3 C) Ruggedness study Influence of the heat-treatment time The Transia Plate Salmonella Gold assay requires a 20 minute-heat inactivation of the enriched RVS broth in order to damage the cell structure and improve the availability of the antigens to the antibodies. This leads also to a sterilisation of the broth which is an important safety factor for the ELISA test. It may happen that the duration of this step is not fully controlled by the user and we found interesting to study the effect of a shortened or increased heat-treatment time on the final response of the assay. In this study heat-treatment times of 15, 20 and 25 minutes were tested; they correspond to the time requested in the package insert, increased or decreased by 5 minutes. This analysis was performed three (3) times, using three (3) different kit lots. 13

14 Methodology Positive strains: From a 20-hour culture at 37 C in BPW (Buffered Peptone Water), two Salmonella strains (Salmonella Enteritidis No and Salmonella Typhimurium No from the Diffchamb collection) were subcultured (0.1 ml into 10 ml) in RVS broth (Rappaport Vassiliadis Soya) for 24 hours at 41.5 C. This culture was then serially diluted and the cell density was estimated, using serial dilutions and plating on TSYE (Tryptone Soya and Yeast Extract Agar), before the heat inactivation at 100 C. Three (3) dilutions were tested by ELISA Negative strain: From a 20-hour culture at 37 C in BPW (Buffered Peptone Water), a non-salmonella strain (Citrobacter freundii No from the Diffchamb collection) was subcultured (0.1 ml into 10 ml) in RVS broth (Rappaport Vassiliadis Soya) for 24 hours at 41.5 C. This culture was then serially diluted and the cell density was estimated, using serial dilutions and plating on TSYE (Tryptone Soya and Yeast Extract Agar), before the heat inactivation at 100 C. The pure culture was tested by ELISA Results The results of this study are summarized in Table 7. They show that the interpretation of the test is the same regardless the heat-treatment time (15, 20 or 25 minutes) Influence of the variation of sample or conjugate volume Another possible deviation that may be introduced by the user includes noncompliance with the specified volumes. In this study, the effects of different volume distribution of sample and conjugate were separately examined Variation of the sample volume The Transia Plate Salmonella Gold assay requires a sample volume of 100 µl. In this study, the effects of a variation of this volume were examined. The negative strain and three (3) dilutions of the positive strains were tested five (5) times, using three (3) different sample volumes: 80, 100 and 120 µl. These volumes correspond respectively to the sample volume requested in the package insert, increased or decreased by 20 µl. This analysis was performed three (3) times, using three (3) different kit lots Methodology Positive Strains: Positive strains: From a 20-hour culture at 37 C in BPW (Buffered Peptone Water), two Salmonella strains (Salmonella Enteritidis No and Salmonella Typhimurium No from the Diffchamb collection) were subcultured (0.1 ml into 14

15 10 ml) in RVS broth (Rappaport Vassiliadis Soya) for 24 hours at 41.5 C. This culture was then serially diluted and the cell density was estimated, using serial dilutions and plating on TSYE (Tryptone Soya and Yeast Extract Agar), before the heat inactivation at 100 C. Three (3) dilutions were tested by ELISA Negative strain: From a 20-hour culture at 37 C in BPW (Buffered Peptone Water), a non-salmonella strain (Citrobacter freundii No from the Diffchamb collection) was subcultured (0.1 ml into 10 ml) in RVS broth (Rappaport Vassiliadis Soya) for 24 hours at 41.5 C. This culture was then serially diluted and the cell density was estimated, using serial dilutions and plating on TSYE (Tryptone Soya and Yeast Extract Agar), before the heat inactivation at 100 C. The pure culture was tested by ELISA Results The results of this study are summarized in Table 8. They show that the interpretation of the test is the same regardless the sample volume (80, 100 or 120 µl) Variation of the conjugate volume The Transia Plate Salmonella Gold assay requires a conjugate volume of 100 µl. In this study, the effects of a variation of this volume were examined.the negative strain and three dilutions of the positive strains were tested five (5) times, using three different conjugate volumes: 80, 100 and 120 µl. These volumes correspond respectively to the conjugate volume requested in the package insert, increased or decreased by 20 µl. This analysis was performed three (3) times, using three (3) different kit lots Methodology Positive strains: From a 20-hour culture at 37 C in BPW (Buffered Peptone Water), two Salmonella strains (Salmonella Enteritidis No and Salmonella Typhimurium No from the Diffchamb collection) were subcultured (0.1 ml into 10 ml) in RVS broth (Rappaport Vassiliadis Soya) for 24 hours at 41.5 C. This culture was then serially diluted and the cell density was estimated, using serial dilutions and plating on TSYE (Tryptone Soya and Yeast Extract Agar), before the heat inactivation at 100 C. Three (3) dilutions were tested by ELISA Negative strain: From a 20-hour culture at 37 C in BPW (Buffered Peptone Water), a non-salmonella strain (Citrobacter freundii No from the Diffchamb collection) was subcultured (0.1 ml into 10 ml) in RVS broth (Rappaport 15

16 Vassiliadis Soya) for 24 hours at 41.5 C. This culture was then serially diluted and the cell density was estimated, using serial dilutions and plating on TSYE (Tryptone Soya and Yeast Extract Agar), before the heat inactivation at 100 C. The pure culture was tested by ELISA Results The results of this study are summarized in Table 9. They show that the interpretation of the test is the same regardless the conjugate volume (80, 100 or 120 µl). 14 Independent Validation Studies AOAC Research Institute approached CCFRA to carry out an independent validation of the Transia Plate Salmonella Gold Test (Diffchamb) against the ISO 6579:2002 method for the detection of Salmonella spp., in two (2) food matrices (raw ground turkey and Brie cheese) Materials and methods The following food matrices were tested: Raw ground turkey Brie cheese Strain details For the raw ground turkey study, a single strain of Salmonella enterica subsp. enterica serovar Enteritidis PT4 (CCFRA code 1004) was used. This strain was isolated from chicken and was obtained from the Central Public Health Laboratory, Colindale, London, United Kingdom. For the Brie cheese study, a single strain of Salmonella enterica subsp. enterica serovar Typhimurium PT195 (CCFRA code 1009) was used. This strain was isolated from milk and was obtained from the Central Public Health Laboratory, Colindale, London, United Kingdom Preparation of inoculum Each strain of Salmonella was cultured in 10ml Nutrient Broth (NB, Oxoid CM1) incubated overnight at 37 o C. Cultures were then serially diluted in Maximum Recovery Diluent (MRD, Lab M Lab103) Inoculation procedure One 1500g portion of each food matrix was inoculated with a dilution of the appropriate culture estimated to give an inoculum level of 1-10 cfu/25g. A further 500g portion of each food remained uninoculated. The inoculated and uninoculated food portions were stored at 2-8 o C for 72h prior to testing. After chilled storage, 20 x 25g samples from the inoculated food portion and 5 x 25g samples from the uninoculated food were weighed out, for each matrix. Also, further samples were taken from the inoculated portion of each 16

17 matrix for MPN analysis (3 x 100g, 3 x 10g, 3 x 1.0g and 3 x 0.1g portions). Samples were then tested by the ISO 6579:2002 method for the detection of Salmonella spp., and by the Transia Plate Salmonella Gold Test, as briefly described overleaf ISO 6579:2002 method for the detection of Salmonella spp. For the pre-enrichment of both food matrices, samples (including MPN samples) were enriched in Buffered Peptone Water (BPW, Oxoid CM1049) and incubated at 37 ± 1 o C for 18 ± 2h (see Appendix 1). Following pre-enrichment, 1ml of each sample was subcultured into 10ml Muller-Kauffmann Tetrathionate-novobiocin broth (MKTTn, Oxoid CM1048) and 0.1ml into 10ml Rappaport-Vassiliadis Soya Peptone broth (RVS, Oxoid CM866). MKTTn broths were incubated at 37 ± 1 o C for 24 ± 3h and RVS broths were incubated at 41.5 ± 0.5 o C for 21-24h. After incubation, a loopful of each selective enrichment broth was streaked onto Xylose Lysine Desoxycholate Agar (XLD, Oxoid CM469) and Brilliant Green Agar (BGA, Oxoid CM263). Plates were incubated at 37 ± 1 o C for 24 ± 3h. Plates were then examined for typical Salmonella colonies. Up-to five (5) typical Salmonella colonies per sample were confirmed by testing for the presence of Salmonella O- and H-antigens by slide agglutination using Salmonella polyvalent O and H antisera (Mast Assure M14294 and M14317) and biochemically using API 20E strips (biomerieux 20100). The MPN of Salmonella present in the initial samples on the day of analysis was determined by referring to the MPN tables in the FDA-BAM manual (8 th Edition) Transia Plate Salmonella Gold test The Transia Plate Salmonella Gold Test was performed on the RVS selective enrichment broths used in the ISO method (see Appendix 1). Following 21-24h incubation in RVS, 1 to 2ml of each sample was taken and heat-treated in a waterbath at 100 o C for 20 minutes. The heat-treated samples were then processed as described in the Transia Plate Salmonella Gold kit insert. Samples found to be assay positive by the Transia Plate Salmonella Gold test, were confirmed by the ISO method, as both were derived from the same 25g sample. Where appropriate, typical Salmonella colonies observed on XLD and BGA plates streaked from unboiled RVS broths were used to confirm both the ISO method and Transia Plate Salmonella gold. 17

18 14.2 Results MPN results The results obtained using the MPN procedure indicated a contamination level in the raw ground turkey to be 6 cfu/25g and in the Brie cheese 2.3 cfu/25g. The 95% confidence intervals on these estimations were and respectively Raw ground turkey The TPSG and ISO results from raw ground turkey are summarized in Table 4 and shown in full in Appendix 20. None of the uninoculated raw ground turkey samples were found to contain Salmonella by the Transia Plate Salmonella Gold test or by the ISO culture method. The TPSG gave equivalent detection rates compared to the ISO culture method for raw ground turkey contaminated with low levels of Salmonella Brie cheese The TPSG and ISO results from Brie cheese are summarized in Table 4 and shown in full in Appendix 21. None of the uninoculated Brie cheese samples were found to contain Salmonella by the Transia Plate Salmonella Gold test or by the ISO culture method. The TPSG gave equivalent detection rates compared to the ISO culture method for Brie cheese contaminated with low levels of Salmonella Conclusion The Transia Plate Salmonella Gold test successfully detected low levels of Salmonella in raw ground turkey and Brie cheese. For both food matrices, the Transia Plate Salmonella Gold test gave the same Salmonella detection rate as the ISO culture method. 15. Discussion In this study, the inclusivity and exclusivity of the Transia Plate Salmonella Gold test were examined by testing the method with pure cultures of various Salmonella and non-salmonella strains. Overall, the test performed very well with inclusivity and exclusivity rates of 96.1 % and 100 %, respectively. The accuracy of the test as compared with the ISO 6579:2002 reference method on 400 samples from 20 matrices performed identically, showing a sensitivity of 99.3 % and a specificity of 97.4 % (see Appendix 22). Despite the low spiking level of the samples (between 1 and 10 CFU/25 g.) the overall agreement between the two (2) methods is excellent with an obtained value of 98.8 %. Transia Plate Salmonella Gold test did not give any false positive signal; when the ELISA test gave a positive or doubtful result, the presumptive test result was then always confirmed, after streaking of the RVS selective broth on selective agars, by the isolation and biochemical confirmation of a Salmonella strain. The ruggedness of Transia Plate Salmonella Gold has been documented by studying the effects of variations in sample or conjugate volume and heat inactivation time. Although important 18

19 variations of these parameters might affect significantly the optical densities, the test interpretation was not modified and the accuracy of the analysis was preserved. 16. Conclusion Our evaluation of the Transia Plate Salmonella Gold demonstrated that this method is equivalent to the ISO 6579:2002 reference method for detection of Salmonella spp. in all foods and feeds tested. This study demonstrated the consistent sensitivity and specificity of the Transia Plate Salmonella Gold assay for the detection of Salmonella spp. in food and feed matrices. 17. References (1) Haeghebaert, S., Le Querrec, F., Vaillant, V., Delarocque Astagneau E. & Bouvet, P. (1998) Les Toxi-Infections Alimentaires Collectives en France en BEH, (2) Anonymous (2000) Preliminary Food Net Data on the Incidence of Foodborne Illnesses Selected Sites, United States. MMWR (Morbidity & Mortality, Weekly Report), 49, (3) FSIS (1999). Salmonella Serotypes Isolated from Raw Meat and Poultry. Electronic memorandum available at (4) U.S. Food and Drug Administration (2003) Bacteriological Analytical Manual Online, January 2001, Chapter 5, Salmonella, (5) Tsang, R.S.W., Schlecht, S., Aleksia, S., Chan, K.H. & Chau P.Y. (1991) Lack of the α-1,2-linked N-acetyl-D-glucosamine Epitope in the Outer Core Structures of Lipopolysaccharides from certain O Serogroups and Subspecies of Salmonella enterica. Res. Microbiolol. 142, (6) Maijala, R., Johansson, T. & Hirn, J. (1992) Growth of Salmonella and Competing Flora in Five Commercial Rappaport-Vassiliadis (RV) Media. Int. J. of Food Microbial 17, 1-8. (7) Peterz, M., Wiberg, C. & Norberg, P. (1989). The effect of Incubation Temperature and Magnesium Chloride Concentration on Growth of Salmonella in Home-Made and in Commercially Available Dehydrated Rappaport-Vassiliadis Broths J. Appl. Bacterial. 66, (8) Owen, P. & Meehan, M. (1994) Immunochemistry of Bacterial Antigens, Immunochemistry, C.J. Van Oss & M.H.V. Van Regenmortel (Eds), Marcel Dekker, New-York, pp

20 (9) Feldsine, P., Abeyta, C. & Andrews W.H. (2002) AOAC International Methods Committee Guidelines for Validation of Qualitative and Quantitative Food Microbiological Official Methods of Analysis, J. AOAC Int. 85, (10) Sharp, J. M. (2002) Development of Immunomagnetic Capture (IMC) Based Techniques for the Detection of Salmonella on Poultry Carcasses, Thesis, Faculty of the Virginia-Maryland Regional College of Veterinary Medicine, pp

21 Table 1 Inclusivity: Salmonella strains Ref. Serotype Group OD Result Ref. Serotype Group OD Result 1716 S. Abaetetuba F 0, S. Cubana G2 0, S. Agama B 0, S. Derby B 0, S. Agona B 1, S. Dublin D1 > S. Alabama D1 1, S. Dublin D1 2, S. Anatum E1 1, S. Dublin D1 2, S. Anatum E1 0, S. Eimsbuttel atypical C4 > S. Antarctica D1 > S. Emek C3 > S. Arizonae II Rough 2, S. Enteritidis D1 1, S. Arizonae IIIa 48 : z4, z23:- Y 0, S. Enteritidis D1 2, S. Arizonae IIIa 48 : z4, z23:- Y 0, S. Enteritidis D1 > S. Arizonae IIIb 61 :-:- non-motile 61 0, S. Enteritidis D1 1, S. Arizonae IIIb 61 :-:- non-motile 61 0, S. Gallinarum pullorum D1 1, S. Arizonae IIIb 61 :-:- non-motile 61 1, S. Give E1 0, S. Arizonae IIIb 61 :i:- 61 1, S. Gloucester B 1, S. Arizonae IIIb 61 :i: z , S. Goldcoast C2 2, S. Arizonae IIIb 61 :k:1,5,7 61 0, S. Goverdham D > S. Arizonae IIIb 61 :r:1,5 61 1, S. Hadar C2 1, S. Babelsberg M 2, S. Hadar C2 > S. Banana B 0, S. Haifa B 1, S. Bergen X 0, S. Havana G2 0, S. Blockley C2 > S. Havana G2 0, S. Bovismorbificans C2 > S. Heidelberg B 1, S. Braenderup C1 > S. Heidelberg B 0, S. Brandenburg B 1, S. Hull I 1, S. Bredeney B 1, S. Hvittingfoss I 2, S. Bredeney B 1, S. I 1,3,19:z27:- E4 0, S. Brunei C3 > S. Idikan G2 0, S. Caracas H 0, S. Indiana B 1, S. Cerro K > S. Indiana B 2, S. Chester B > S. Infantis C1 1, S. Coeln C1 2, S. Infantis C1 1, S. Corvallis C3 1, S. Johannesburg R 0,

22 Table 1 Inclusivity: Salmonella strains (continued) Ref. Serotype Group OD Result Ref. Serotype Group OD Result 715 S. Kapemba D1 2, S. Poona G1 2, S. Kedougou G2 0, S. Quentin D2 1, S. Kedougou G2 1, S. Ramatgan N 0, S. Kentucky C3 2, S. Reading B 2, S. Kentucky C3 1, S. Rissen C1 1, S. Koketime V 0, S. Saint-Paul B 1, S. Kottbus C2 1, S. Saint-Paul B 1, S. Lagos B 1, S. Salford I 0, S. Lille C1 2, S. Schleissheim B 1, S. Livingstone C1 0, S. Schwarzengrund B 1, S. London E1 0, S. Senegal F 0, S. Manhattan C3 1, S. Senftenberg E4 0, S. Mbandaka C1 1, S. Senftenberg E4 1, S. Mbandaka C1 1, S. Senfentberg E4 1, S. Meleagridis E1 0, S. Stanleyville B 1, S. Mikawasima C1 1, S. Stuivenberg E4 0, S. Minnesota L 0, S. Tennesse C1 1, S. Montevideo C1 1, S. Thompson C1 2, S. Montevideo C1 1, S. Tripoli B 1, S. Muenchen C2 1, S. Typhi D1 2, S. Newport C2 1, S. Typhimurium B 1, S. Newport C2 > S. Typhimurium B 1, S. Nima M 0, S. Typhimurium B 1, S. Norton C1 2, S. Typhimurium B 1, S. Ohio C1 1, S. Urbana N 0, S. Oranienburg C1 1, S. Veneziana F 1, S. Orion E1 0, S. Virchow C1 2, S. Ouakam D2 1, S. Virchow C1 1, S. Panama D1 1, S. Virginia C3 1, S. paratyphi A A 2, S. Wien B 1, S. paratyphi B B 1, S. Zanzibar E1 2, S. paratyphi B B 1, Sal. I 47 : z4, z23 : - X 0, S. Poona G1 2,

23 Table 2 Exclusivity: Non-Salmonella strains Ref. Bacteria species OD Result Ref. Bacteria species OD Result 567 Aeromonas hydrophila 0, E. coli O111 0, Bac. Cereus 0, E. coli O126 0, Bac. Cereus 0, E. coli O157 0, Bac. sphaericus 0, E. coli O157 H7 0,082 - BA16* Bac. licheniformis 0, E. coli O26 H11 0,081 - LE3* Candida albicans 0,069 - ESC15* E. Hermanii 0, Cit. amalonaticus 0, Hafnia alvei 0, Cit. braakii 0, Hafnia alvei 0, Cit. braakii 0, Klebsiella oxytoca 0, Cit.braakii 0, Klebsiella pneumoniae 0, Cit.braakii 0, Klebsiella pneumoniae 0, Cit.braakii 0, List. monocytogenes 0, Cit. diversus 0, Proteus mirabilis 0, Cit. diversus 0, Proteus morganii 0, Cit. freundii 0, Proteus vulgaris 0, Cit. freundii 0, Pseudo. aeruginosa 0, Cit. freundii 0, Pseudo. fluorescens 0, Cit. freundii 0,080 - LE6* Rhodotorula rubra 0, Cit. freundii 0,102 - LE5* Saccharomyces cerevisiae 0, Cit. freundii 0, Serratia liquefaciens 0, Cit. freundii 0, Serratia marcescens 0, Cit. freundii 0, Shigella flexneri 0, Cit. youngae 0,046 - SHI16* Shigella sonnei 0, Cit. youngae 0, Staph. aureus 0, Ent. agglomerans 0, Staph. aureus 0, Ent. Amnigenus 0, Staph. aureus 0, Ent. cancerogenus 0, Staph. aureus A + 0, Ent. cloacae 0, Staph. aureus A + 0, Ent. cloacae 0, Staph. epidermis 0, Ent. cloacae 0, Staph. haemoliticus 0, Ent. intermedium 0, Staph. hominis 0, Ent. nimipressuralis 0,068 - ST12* Staph. hyicus 0, Ent. sakazakii 0,081 - ST6* Staph. Saprophyticus 0,057-17* Erwinia spp. 0, Yersinia enterocolitica 0, Escherichia adecarboxylata 0, Yersinia enterocolitica 0, E. coli 0, Yersinia pseudotuberculosis 0,071 - * reference from Pasteur Institute Lille (SERMHA) 23

24 Table 3 - Inclusivity/Exclusivity Study Results Strains TPSG results Total Positive Negative Salmonella Non-Salmonella Total Inclusivity = 124 / 129 = 96.1 % Exclusivity = 72 / 72 = % 24

25 Table 4 : Summary results comparing the detection of Salmonella by the TPSG and ISO methods Test sample Level Positive with Positive Sensivity b False negative c, Specificity d, False Total No., % MPN TP Salmonella Gold with % % positive e, % of test χ² a / 25 g ISO ISO ISO portions Presumptive Confirmed TPSG TPSG TPSG TPSG Agreement % Cooked chicken Low f Control f Cantaloupe Low f Control f Sausages Low f Control f Raw shrimps Low f Control f Yogurt Low f Control f Mayonnaise Low f Control f Shell eggs Low f Control f Frozen berries Low f Control f Bean sprouts Low f Control f Raw ground beef Low f Control f Smoked fish Low f Control f Uncooked noodles Low f Control f Milk chocolate Low f Control f Black pepper Low f Control f Cake mix Low f Control f Infant formula Low f Control f Dry pet food Low f Control f Raw milk Low Control f Raw ground turkey g Low f Control f Brie cheese g Low f Control f