Transia Plate Salmonella Gold for the detection of ... Plate_Sal_AOAC PTM... · Transia Plate...

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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, 69007 Lyon, France Submitting Company Diffchamb AB, F O Petersons Gata 32, SE-421 31 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)

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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.

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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 29483 USA Phone +843 873 3007 (ext. 7103) or 1-866 -DIFFCHAMB Fax +843 873 3545 [email protected] Website address: www.diffchamb.com

From abroad: Diffchamb AB F O Petersons Gata 32 421 31 Västra Frölunda Sweden Phone +46 31 742 33 50 Fax +46 31 58 33 70 [email protected]

5.4 Test Kit Reagents:

5.4.1 Negative control – ready to use – 1 x 6 mL (SA0180), 2 x 15 mL (SA0190). 5.4.2 Positive control: LPS (lipopolysaccharide) – ready to use – 1 x 3 mL

(SA0180), 2 x 8 mL (SA0190). 5.4.3 Conjugate: anti-Salmonella antibodies conjugated to peroxydase – ready to

use – 1 x 15 mL (SA0180), 4 x 30 mL (SA0190). 5.4.4 Washing buffer – concentrated 20X – 1 x 60 mL (SA0180), 1 x 800 mL

(SA0190). 5.4.5 Substrate: Urea-H2O2 – 1 x 10 mL (SA0180), 3 x 30 mL (SA0190). 5.4.6 Chromogen: TMB (Tetramethylbenzidine) – 1 x 10 mL (SA0180), 3 x 30 mL

(SA0190).

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5.4.7 Stop solution: H2SO4 – ready to use – 1 x 15 mL (SA0180), 5 x 30 mL (SA0190).

6. Additional Media and Reagents

6.1 Enrichment: 6.1.1 Distilled water. 6.1.2 Buffered peptone water. 6.1.3 Rappaport-Vassiliadis Soya broth (RVS).

6.2 Confirmation of positive results: 6.2.1 Isolation: Selective agar plate such as xylose lysine deoxycholate (XLD) or

brilliant green agar (BGA). 6.2.2 Identification: Biochemical identification gallery (such as API 20E,

BioMérieux, Art. No. 20100 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. 7.10 Water bath 100°C (boiling water). 7.11 Micropipette 100-1000 µL.

7.12 Micropipette 10-100 µL (optional). 7.13 Wash bottle (or preferably an automatic microplate washer). 7.14 Absorbent paper. 7.15 Multipipette with 2.5 and 5 mL tips.

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7.16 Microplate reader – single or double beam reading 450 nm filter and reference filter ≥595 nm.

7.17 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, 1187-1200.

9. 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.

10.2 Incubate at 37 ± 1°C for 16-20 hours. 10.3 Homogenize and inoculate 0.1 mL of the pre-culture broth in 10 mL of RVS. 10.4 Incubate for 18-24 hours at 41.5 ± 0.5°C. 10.5 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.

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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.

11.2 Distribute 100 µL of the controls and the samples into the assigned wells. Cover the

plate.

11.3 Incubate at room temperature (18-30°C) for one hour. Prepare the washing buffer. 11.4 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.

11.5 Add 100 µL of conjugate (vial 4) to each well. Be careful not to touch the wells with

the dispensing tip. Cover the plate. 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]. 11.7 Wash the microplate 5 times as indicated in paragraph 11.4. 11.8 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.

Controls and samples

Conjugate

Substrate/chromogen mixture

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11.9 Incubate at room temperature (18-30°C) for 15 minutes. 11.10 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.

11.11 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 0.700. The optical density of the negative control (NC) must be lower than or equal to 0.150

(for double beam reading) or 0.200 (for single beam reading). If the controls do not meet these requirements, the results are invalid. 12.2 Positive threshold The positive threshold is calculated as the average of the negative controls plus 0.11: [(NC1 + NC2) / 2] + 0.11 12.3 Negative threshold The negative threshold is calculated as the positive threshold multiplied by 0.9 12.4 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).

Stop solution

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13 Internal Validation Studies Internal validation studies were conducted at the R&D Centre of the Diffchamb Group (Diffchamb S.A. – Lyon, France). 13.1 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).

13.1.1 Methodology From a 20-hour culture in BPW (buffered peptone water) at 37 ± 1oC, each

Salmonella strain was subcultured (0.1 mL into 10 mL) in RVS (Rappaport Vassiliadis soya broth) at 41.5 ± 0.5oC for 18-24 hours. Following enrichment, 1 mL of each broth was taken and heat-treated in a water bath at 100oC 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 ± 1oC for 24 ± 3 h.

13.1.2 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 %.

13.2 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.

13.2.1 Methodology

From a 20-hour culture in BPW (buffered peptone water) at 37 ± 1oC, each non-Salmonella strain was subcultured (0.1 mL into 10 mL) in BPW at 37 ± 1oC for 18-24 hours. Following enrichment, 1 mL of each broth was taken and heat-treated in a water bath at 100oC 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 ± 1oC for 24 ± 3 h.

13.2.2 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 %.

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13.3 Method Comparison Study

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. 13.3.1 Methodology

13.3.1.1 Selected strains

As agreed with AOAC R.I. the following combinations matrix/strain was tested:

Matrix Total count (cfu/g)

Strain Reference Origin

Cooked chicken 2 x 104 Salmonella Kentucky 305 AFSSA – France

Raw milk 5.4 x 106 Salmonella Dublin 1236 Dairy – Denmark

Cantaloupe 3.5 x 105 Salmonella Poona 781 Pasteur Institute Paris – France

Sausages 4.4 x 107 Salmonella Bredeney 1656 Pasteur Institute Lille – France

Raw shrimps 8.1 x 108 Salmonella Montevideo 1449 NCTC 5747

Yogurt 1 x 102 Salmonella Typhimurium 1654 Pasteur Institute Lille – France

Mayonnaise < 102 Salmonella Heidelberg 1689 Isolated from chicken gizzard – France

Shell eggs 2.7 x 104 Salmonella Enteritidis 1535 Isolated from cocoa - France

Frozen red berries & currants 2 x 103 Salmonella Newport 1442 Isolated from chicken wings – France

Bean sprouts 4.5 x 108 Salmonella Infantis 1305 Service laboratory – Australia

Raw ground beef 1.5 x 109 Salmonella Anatum 1622 Service laboratory – UK

Smoked fish (trout) 6.9 x 107 Salmonella Virchow 1451 Isolated from chicken filet – France

Fresh pasta < 102 Salmonella IIIb 61:r:1.5 1575 Isolated from lamb tongue – France

Milk chocolate 1.2 x 102 Salmonella Senftenberg 1486 Dairy – UK

Ground black pepper 6.6 x 104 Salmonella Indiana 1428 Isolated from cow liver – France

Cake mix 8.2 x 103 Salmonella Thompson 306 Food Inspection Services – France

Dry milk-based infant formula < 102 Salmonella Blockley 1100 Isolated from chicken gizzard – France

Dry food for cat 7 x 102 Salmonella Mbandaka 1618 Service laboratory – UK

13.3.1.2 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.

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For dry foods contamination, a freeze-dried bacterial culture was grinded to a fine powder and added to 200-500 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.

13.3.1.3 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 10-50 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 48-72 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.

13.3.1.4 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).

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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 (8th edition).

13.3.1.5 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 heat-treated 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).

13.3.2 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.

13.3.3 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).

13.4 Lot-to-lot and Stability Study

13.4.1 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.

13.4.1.1 Methodology Positive test strains: From a 24-hour culture at 37 ± 2º C in BPW (Buffered Peptone Water), each Salmonella strain (No. 1431 and 1435 from the Diffchamb collection) was subcultured (0.2 mL into 10 mL) in

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RVS (Rappaport Vassiliadis Soya broth) for 24 h ± 2 hours at 41.5 ± 0.5oC. 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 (1x105, 5x105 and 1x106 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 ± 0.5oC. The cell count was then estimated by reading the optical density at 470 nm and the cultures were adjusted to 109 cells/mL by dilution in BPW before a heat inactivation of 20 minutes at 100°C.

13.4.1.2 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 1x105 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 1x105 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.

13.4.2. 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.

13.4.2.1. Methodology

Positive test strains: From a 24-hour culture at 37 ± 2º C in BPW (Buffered Peptone Water), each Salmonella strain (No. 1305 and 1435 from the Diffchamb collection) was subcultured (0.2 mL into 10 mL) in

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RVS (Rappaport Vassiliadis Soya broth) for 24 h ± 2 hours at 41.5 ± 0.5oC. 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. 1163 from the Diffchamb collection) was subcultured (0.2 mL into 10 mL) in BPW for 24 h ± 2 hours at 41.5 ± 0.5oC. The cell count was then estimated by reading the optical density at 470 nm and the cultures were adjusted to 109 cells/mL by dilution in RVS before a heat inactivation of 20 minutes at 100°C.

13.4.2.2. 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 oC. 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).

13.5 Ruggedness study

13.5.1 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.

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13.5.1.1 Methodology

13.5.1.1.1. Positive strains: From a 20-hour culture at 37°C in BPW (Buffered Peptone Water), two Salmonella strains (Salmonella Enteritidis No. 1834 and Salmonella Typhimurium No. 1654 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.

13.5.1.1.2. Negative strain: From a 20-hour culture at 37°C in BPW (Buffered

Peptone Water), a non-Salmonella strain (Citrobacter freundii No. 1694 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.

13.5.1.2 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).

13.5.2. Influence of the variation of sample or conjugate volume Another possible deviation that may be introduced by the user includes non-compliance with the specified volumes. In this study, the effects of different volume distribution of sample and conjugate were separately examined.

13.5.2.1. 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.

13.5.2.1.1. Methodology

13.5.2.1.1.1. Positive Strains: Positive strains: From a 20-hour culture at 37°C in BPW (Buffered Peptone Water), two Salmonella strains (Salmonella Enteritidis No. 1834 and Salmonella Typhimurium No. 1654 from the Diffchamb collection) were subcultured (0.1 mL into

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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.

13.5.2.1.1.2. Negative strain:

From a 20-hour culture at 37°C in BPW (Buffered Peptone Water), a non-Salmonella strain (Citrobacter freundii No. 1694 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.

13.5.2.1.2. 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).

13.5.2.2. 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.

13.5.2.2.1. Methodology

13.5.2.2.1.1. Positive strains: From a 20-hour culture at 37°C in BPW (Buffered Peptone Water), two Salmonella strains (Salmonella Enteritidis No. 1834 and Salmonella Typhimurium No. 1654 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.

13.5.2.2.1.2. Negative strain: From a 20-hour culture at 37°C in BPW

(Buffered Peptone Water), a non-Salmonella strain (Citrobacter freundii No. 1694 from the Diffchamb collection) was subcultured (0.1 mL into 10 mL) in RVS broth (Rappaport

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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.

13.5.2.2.2. 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).

14.1. Materials and methods The following food matrices were tested: Raw ground turkey Brie cheese 14.1.1 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.

14.1.2 Preparation of inoculum Each strain of Salmonella was cultured in 10ml Nutrient Broth (NB, Oxoid CM1) incubated overnight at 37oC. Cultures were then serially diluted in Maximum Recovery Diluent (MRD, Lab M Lab103).

14.1.3 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 2-8oC 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

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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.

14.1.4 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 ± 1oC 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 ± 1oC for 24 ± 3h and RVS broths were incubated at 41.5 ± 0.5oC 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 ± 1oC 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 (8th Edition).

14.1.5 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 100oC 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.

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14.2 Results

14.2.1 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 1.45-24.875 and 0.55-9.725 respectively.

14.2.2 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.

14.2.3 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.

14.3 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

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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 1997. BEH, 177-81

(2) Anonymous (2000) Preliminary Food Net Data on the Incidence of Foodborne

Illnesses Selected Sites, United States. MMWR (Morbidity & Mortality, Weekly Report), 49, 201-205

(3) FSIS (1999). Salmonella Serotypes Isolated from Raw Meat and Poultry.

Electronic memorandum available at www.fsis.usda.gov/OPHS/haccp/serolyr.htm (4) U.S. Food and Drug Administration (2003) Bacteriological Analytical Manual

Online, January 2001, Chapter 5, Salmonella, http://www.cfsan.fda.gov/~comm/bam-5.html

(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, 521-533.

(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, 523-528

(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 393-453.

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(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, 1187-1200.

(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 3-5.

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Table 1 – Inclusivity: Salmonella strains

Ref. Serotype Group OD Result Ref. Serotype Group OD Result

1716 S. Abaetetuba F 0,541 + 1738 S. Cubana G2 0,475 +

1623 S. Agama B 0,841 + 1692 S. Derby B 0,543 +

1607 S. Agona B 1,811 + 1236 S. Dublin D1 >3 +

712 S. Alabama D1 1,963 + 1604 S. Dublin D1 2,457 +

1622 S. Anatum E1 1,215 + 1466 S. Dublin D1 2,375 +

1685 S. Anatum E1 0,455 + 332 S. Eimsbuttel atypical C4 >3 +

713 S. Antarctica D1 >3 + 1478 S. Emek C3 >3 +

1483 S. Arizonae II Rough 2,185 + 688 S. Enteritidis D1 1,771 +

787 S. Arizonae IIIa 48 : z4, z23:- Y 0,056 - 1195 S. Enteritidis D1 2,013 +

1655 S. Arizonae IIIa 48 : z4, z23:- Y 0,071 - 1214 S. Enteritidis D1 >3 +

1148 S. Arizonae IIIb 61 :-:- non-motile 61 0,803 + 1535 S. Enteritidis D1 1,751 +

1329 S. Arizonae IIIb 61 :-:- non-motile 61 0,505 + 668 S. Gallinarum pullorum D1 1,942 +

1117 S. Arizonae IIIb 61 :-:- non-motile 61 1,155 + 1609 S. Give E1 0,748 +

1308 S. Arizonae IIIb 61 :i:- 61 1,103 + 299 S. Gloucester B 1,851 +

1273 S. Arizonae IIIb 61 :i: z53 61 1,134 + 1487 S. Goldcoast C2 2,120 +

1591 S. Arizonae IIIb 61 :k:1,5,7 61 0,909 + 1475 S. Goverdham D >3 +

1575 S. Arizonae IIIb 61 :r:1,5 61 1,170 + 1453 S. Hadar C2 1,816 +

1481 S. Babelsberg M 2,407 + 1458 S. Hadar C2 >3 +

1691 S. Banana B 0,543 + 1482 S. Haifa B 1,951 +

1644 S. Bergen X 0,061 - 1621 S. Havana G2 0,719 +

1100 S. Blockley C2 >3 + 1695 S. Havana G2 0,613 +

1444 S. Bovismorbificans C2 >3 + 1431 S. Heidelberg B 1,147 +

708 S. Braenderup C1 >3 + 1689 S. Heidelberg B 0,915 +

1351 S. Brandenburg B 1,527 + 1473 S. Hull I 1,842 +

1283 S. Bredeney B 1,453 + 1467 S. Hvittingfoss I 2,168 +

1656 S. Bredeney B 1,668 + 1663 S. I 1,3,19:z27:- E4 0,543 +

1474 S. Brunei C3 >3 + 836 S. Idikan G2 0,651 +

1737 S. Caracas H 0,634 + 1381 S. Indiana B 1,996 +

1129 S. Cerro K >3 + 1428 S. Indiana B 2,327 +

862 S. Chester B >3 + 1305 S. Infantis C1 1,636 +

1251 S. Coeln C1 2,335 + 1306 S. Infantis C1 1,232 +

1066 S. Corvallis C3 1,192 + 1476 S. Johannesburg R 0,595 +

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Table 1 – Inclusivity: Salmonella strains (continued)

Ref. Serotype Group OD Result Ref. Serotype Group OD Result

715 S. Kapemba D1 2,097 + 1241 S. Poona G1 2,075 +

1111 S. Kedougou G2 0,841 + 838 S. Quentin D2 1,733 +

1613 S. Kedougou G2 1,441 + 311 S. Ramatgan N 0,668 +

305 S. Kentucky C3 2,034 + 919 S. Reading B 2,154 +

1617 S. Kentucky C3 1,574 + 1238 S. Rissen C1 1,478 +

1477 S. Koketime V 0,535 + 1412 S. Saint-Paul B 1,299 +

1471 S. Kottbus C2 1,893 + 1415 S. Saint-Paul B 1,313 +

736 S. Lagos B 1,645 + 1698 S. Salford I 0,412 +

296 S. Lille C1 2,006 + 1479 S. Schleissheim B 1,651 +

1645 S. Livingstone C1 0,626 + 900 S. Schwarzengrund B 1,670 +

859 S. London E1 0,968 + 1697 S. Senegal F 0,582 +

1560 S. Manhattan C3 1,417 + 888 S. Senftenberg E4 0,735 +

1618 S. Mbandaka C1 1,557 + 1406 S. Senftenberg E4 1,046 +

1746 S. Mbandaka C1 1,049 + 1486 S. Senfentberg E4 1,421 +

1261 S. Meleagridis E1 0,427 + 904 S. Stanleyville B 1,645 +

1612 S. Mikawasima C1 1,604 + 702 S. Stuivenberg E4 0,525 +

303 S. Minnesota L 0,913 + 1276 S. Tennesse C1 1,553 +

1213 S. Montevideo C1 1,461 + 306 S. Thompson C1 2,117 +

1449 S. Montevideo C1 1,219 + 763 S. Tripoli B 1,023 +

1328 S. Muenchen C2 1,722 + 730 S. Typhi D1 2,383 +

1210 S. Newport C2 1,926 + 293 S. Typhimurium B 1,698 +

1442 S. Newport C2 >3 + 924 S. Typhimurium B 1,600 +

783 S. Nima M 0,484 + 1024 S. Typhimurium B 1,189 +

1614 S. Norton C1 2,337 + 1654 S. Typhimurium B 1,448 +

1207 S. Ohio C1 1,744 + 784 S. Urbana N 0,104 -

1188 S. Oranienburg C1 1,259 + 780 S. Veneziana F 1,420 +

1701 S. Orion E1 0,557 + 1194 S. Virchow C1 2,033 +

837 S. Ouakam D2 1,542 + 1451 S. Virchow C1 1,548 +

1159 S. Panama D1 1,790 + 1468 S. Virginia C3 1,411 +

731 S. paratyphi A A 2,015 + 681 S. Wien B 1,732 +

732 S. paratyphi B B 1,318 + 295 S. Zanzibar E1 2,123 +

1002 S. paratyphi B B 1,287 + 786 Sal. I 47 : z4, z23 : - X 0,037 -

781 S. Poona G1 2,533 +

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Table 2 – Exclusivity: Non-Salmonella strains

Ref. Bacteria species OD Result Ref. Bacteria species OD Result 567 Aeromonas hydrophila 0,064 - 1183 E. coli O111 0,107 - 641 Bac. Cereus 0,077 - 1184 E. coli O126 0,083 - 664 Bac. Cereus 0,085 - 1404 E. coli O157 0,081 - 383 Bac. sphaericus 0,077 - 1536 E. coli O157 H7 0,082 -

BA16* Bac. licheniformis 0,072 - 1156 E. coli O26 H11 0,081 - LE3* Candida albicans 0,069 - ESC15* E. Hermanii 0,053 - 735 Cit. amalonaticus 0,077 - 1391 Hafnia alvei 0,083 -

1409 Cit. braakii 0,081 - 1414 Hafnia alvei 0,082 - 1677 Cit. braakii 0,039 - 451 Klebsiella oxytoca 0,083 - 433 Cit.braakii 0,043 - 1079 Klebsiella pneumoniae 0,080 - 433 Cit.braakii 0,057 - 1162 Klebsiella pneumoniae 0,084 - 795 Cit.braakii 0,041 - 1507 List. monocytogenes 0,085 - 933 Cit. diversus 0,078 - 926 Proteus mirabilis 0,079 -

1163 Cit. diversus 0,077 - 436 Proteus morganii 0,062 - 1082 Cit. freundii 0,038 - 917 Proteus vulgaris 0,066 - 1082 Cit. freundii 0,055 - 133 Pseudo. aeruginosa 0,072 - 1085 Cit. freundii 0,039 - 522 Pseudo. fluorescens 0,076 - 1289 Cit. freundii 0,080 - LE6* Rhodotorula rubra 0,096 - 1348 Cit. freundii 0,102 - LE5* Saccharomyces cerevisiae 0,079 - 1438 Cit. freundii 0,080 - 927 Serratia liquefaciens 0,067 - 1043 Cit. freundii 0,079 - 429 Serratia marcescens 0,078 - 1694 Cit. freundii 0,086 - 328 Shigella flexneri 0,082 - 1660 Cit. youngae 0,046 - SHI16* Shigella sonnei 0,085 - 1703 Cit. youngae 0,047 - 572 Staph. aureus 0,059 - 815 Ent. agglomerans 0,068 - 574 Staph. aureus 0,076 - 764 Ent. Amnigenus 0,071 - 1069 Staph. aureus 0,080 - 937 Ent. cancerogenus 0,079 - 1132 Staph. aureus A + 0,077 - 769 Ent. cloacae 0,084 - 1138 Staph. aureus A + 0,084 - 427 Ent. cloacae 0,065 - 637 Staph. epidermis 0,079 -

1699 Ent. cloacae 0,083 - 588 Staph. haemoliticus 0,067 - 1078 Ent. intermedium 0,078 - 852 Staph. hominis 0,064 - 1693 Ent. nimipressuralis 0,068 - ST12* Staph. hyicus 0,080 - 839 Ent. sakazakii 0,081 - ST6* Staph. Saprophyticus 0,057 - 17* Erwinia spp. 0,074 - 570 Yersinia enterocolitica 0,080 - 765 Escherichia adecarboxylata 0,068 - 428 Yersinia enterocolitica 0,079 -

1702 E. coli 0,048 - 430 Yersinia pseudotuberculosis 0,071 -

* reference from Pasteur Institute Lille (SERMHA)

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Table 3 - Inclusivity/Exclusivity Study Results

Strains TPSG results Total

Positive Negative

Salmonella 124 5 129

Non-Salmonella 0 72 72

Total 124 77 201 Inclusivity = 124 / 129 = 96.1 % Exclusivity = 72 / 72 = 100.0 %

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Table 4 : Summary results comparing the detection of Salmonella by the TPSG and ISO methods Positive with

TP Salmonella Gold Sensivityb, % False negativec, %

Specificityd, %

False positivee, % Test sample Level MPN

/ 25 g

Total No. of test

portions Presumptive Confirmed

Positive with ISO 6579

χ²a TPSG ISO

6579 TPSG ISO 6579 TPSG TPSG

Agreement %

Low 6 20 16 16 16 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Cooked chicken Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 7 20 19 19 19 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Cantaloupe Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 4 20 18 18 18 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Sausages Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 6 20 15 15 15 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Raw shrimps Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 1 20 14 14 14 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Yogurt Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 1 20 6 6 6 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Mayonnaise Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 1 20 13 13 13 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Shell eggs Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 4 20 13 13 13 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Frozen berries Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 1 20 17 17 17 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Bean sprouts Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 2 20 17 17 17 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Raw ground beef Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 2 20 14 14 14 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Smoked fish Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 1 20 19 19 19 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Uncooked noodles Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 2 20 17 17 17 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Milk chocolate Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 10 20 14 14 14 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Black pepper Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 6 20 19 19 19 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Cake mix Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 1 20 10 10 10 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Infant formula Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 2 20 11 11 11 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Dry pet food Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 1 20 6 6 5 0 75.0 62.5 25.0 37.5 80.0 20.0 75.0 Raw milk Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 6 20 17 17 17 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Raw ground turkeyg Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0 Low 2 20 12 12 12 -f 100.0 100.0 0.0 0.0 100.0 0.0 100.0 Brie cheeseg Control 0 5 0 0 0 -f - - - - 100.0 0.0 100.0

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a χ² ,as defined by McNemar is (|a - b| - 1)² / (a + b) where a = test portions positive by TPSG and negative by ISO method and b = test portions negative by TPSG and positive by ISO method. A χ² value greater than 3.84 indicated significance at P < 0.05.

b Sensitivity rate was defined as 100 times the total number of analyzed positive test portions among “known” positive tests portions divided by the total number of “known” test portions, where “known” positive was defined as test portions confirmed positive by the reference method.

c Incidence of false negatives was 100 – sensitivity rate. d Specificity rate was defined as 100 times the total number of analyzed negative test portions among “known” negative test portions divided by

the total number of “known” negative test portions, where known negative was defined as test portions confirmed negative by the reference method and negative controls.

e Incidence of false positives was 100 – specificity rate. f Statistical analysis was not applicable, methods gave equivalent results. g Matrix included in the independent validation studies.

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Table 5 – Lot-to-lot consistency study

Release date 10 MAY 04 06 APR 04 26 NOV 03

Lot 21A 12A 58Z

Period of testing Release (newly manufactured) Release + 40 days Release + 9 months

Test parameters

NC1 : 0.050 NC2 : 0.051 PC : 2.820

Negative threshold : 0.144 Positive threshold : 0.160

NC1 : 0.040 NC2 : 0.042 PC : 2.148


NC1 : 0.052 NC2 : 0.051 PC : 2.181


Salmonella Heidelberg ( strain 1431) 1x105 cells/mL 0.132 (-) 0.138 (-) 0.087 (-) 0.095 (-) 0.080 (-) 0.084 (-) 5x105 cells/mL 0.320 (+) 0.322 (+) 0.234 (+) 0.227 (+) 0.203 (+) 0.211 (+) 1x106 cells/mL 0.536 (+) 0.523 (+) 0.400 (+) 0.402 (+) 0.426 (+) 0.416 (+) Salmonella Enteritidis ( strain 1435) 1x105 cells/mL 0.161 (+) 0.165 (+) 0.156 (+) 0.165 (+) 0.163 (+) 0.174 (+) 5x105 cells/mL 0.480 (+) 0.461 (+) 0.391 (+) 0.397 (+) 0.387 (+) 0.391 (+) 1x106 cells/mL 0.827 (+) 0.837 (+) 0.617 (+) 0.601 (+) 0.573 (+) 0.569 (+) Enterobacter nimipressuralis ( strain 1693) 109 cells/mL 0.057 (-) 0.061 (-) 0.044 (-) 0.042 (-) 0.052 (-) 0.058 (-) Citrobacter freundii ( strain 1694) 109 cells/mL 0.064 (-) 0.052 (-) 0.041 (-) 0.053 (-) 0.061 (-) 0.053 (-) Escherichia coli (strain 1771) 109 cells/mL 0.059 (-) 0.061 (-) 0.048 (-) 0.050 (-) 0.056 (-) 0.058 (-)

NC: Negative control (-): Negative interpretation PC: Positive control (+): Positive interpretation

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Table 6 – Stability

Lot 26W – Release date : 26 JUL 2004

Period of testing Release (newly manufactured)

Release + 14 days at 5 ± 3oC


Test parameters

NC1 : 0.056 NC2 : 0.055 PC : 2.426


NC1 : 0.046 NC2 : 0.049 PC : 2.179


NC1 : 0.079 NC2 : 0.081 PC : 1.279


Salmonella Infantis ( strain 1305) 1x106 cells/mL 0.219 (+) 0.224 (+) 0.178 (+) 0.178 (+) 0.205 (+) 0.212 (+) 2.5x106 cells/mL 0.356 (+) 0.366 (+) 0.295 (+) 0.289 (+) 0.355 (+) 0.347 (+) 5x106 cells/mL 0.496 (+) 0.502 (+) 0.468 (+) 0.472 (+) 0.560 (+) 0.595 (+) Salmonella Enteritidis ( strain 1435) 1x105 cells/mL 0.220 (+) 0.211 (+) 0.193 (+) 0.194 (+) 0.200 (+) 0.197 (+) 5x105 cells/mL 0.679 (+) 0.664 (+) 0.605 (+) 0.603 (+) 0.597 (+) 0.607 (+) 1x106 cells/mL 1.048 (+) 1.109 (+) 0.937 (+) 0.926 (+) 0.963 (+) 0.935 (+) Citrobacter diversus ( strain 1163) 109 cells/mL 0.071 (-) 0.075 (-) 0.051 (-) 0.052 (-) 0.090 (-) 0.093 (-)


Lot 26W – Release date : 26 JUL 2004

Period of testing Release (newly manufactured)



Test parameters

NC1 : 0.056 NC2 : 0.055 PC : 2.426


NC1 : 0.052 NC2 : 0.055 PC : 2.571


NC1 : 0.089 NC2 : 0.085 PC : 1.080


Salmonella Infantis ( strain 1305) 1x106 cells/mL 0.219 (+) 0.224 (+) 0.177 (+) 0.186 (+) 0.210 (+) 0.204 (+) 2.5x106 cells/mL 0.356 (+) 0.366 (+) 0.296 (+) 0.309 (+) 0.326 (+) 0.326 (+) 5x106 cells/mL 0.496 (+) 0.502 (+) 0.457 (+) 0.463 (+) 0.424 (+) 0.438 (+) Salmonella Enteritidis ( strain 1435) 1x105 cells/mL 0.220 (+) 0.211 (+) 0.187 (+) 0.183 (+) 0.204 (+) 0.209 (+) 5x105 cells/mL 0.679 (+) 0.664 (+) 0.553 (+) 0.555 (+) 0.527 (+) 0.523 (+) 1x106 cells/mL 1.048 (+) 1.109 (+) 0.884 (+) 0.906 (+) 0.885 (+) 0.866 (+) Citrobacter diversus ( strain 1163) 109 cells/mL 0.071 (-) 0.075 (-) 0.059 (-) 0.060 (-) 0.097 (-) 0.097 (-)


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Table 7 - Ruggedness Study - Variation of the heat treatment time

Lot 2A

Heat treatment time 15 minutes 20 minutes 25 minutes

Test parameters

NC1 : 0.077 NC2 : 0.077 PC : 1.968

NEG threshold: 0.168 POS threshold: 0.187

NC1 : 0.082 NC2 : 0.085

PC : 2.19 NEG threshold: 0.174 POS threshold: 0.194

NC1 : 0.083 NC2 : 0.083 PC : 2.207


S. Typhimurium ( strain 1654) 2.5x105 cells/mL 0.133 (-) 0.141 (-) 0.144 (-) 2.5x106 cells/mL 0.359 (+) 0.355 (+) 0.384 (+) 2.5x107 cells/mL 0.912 (+) 0.873 (+) 0.896 (+) S. Enteritidis ( strain 1834) 7.6x103 cells/mL 0.115 (-) 0.124 (-) 0.123 (-) 7.6x104 cells/mL 0.275 (+) 0.344 (+) 0.376 (+) 7.6x105 cells/mL 1.194 (+) 1.278 (+) 1.423 (+) Citrobacter freundii (strain 1694) 109 cells/mL 0.088 (-) 0.096 (-) 0.093 (-)

NC: Negative control (-): Negative interpretation PC: Positive control (+): Positive interpretation O.D. written in Italic are average values of 5 replicates

Lot 8A


Test parameters

NC1 : 0.077 NC2 : 0.079 PC : 2.624


NC1 : 0.100 NC2 : 0.096 PC : 2.802


NC1 : 0.089 NC2 : 0.083 PC : 2.661




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Lot 12A


Test parameters

NC1 : 0.113 NC2 : 0.103 PC : 3.181


NC1 : 0.092 NC2 : 0.091 PC : 2.870


NC1 : 0.096 NC2 : 0.100 PC : 2.897




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Table 8 - Ruggedness Study - Variation of the sample volume

Lot 2A

Sample volume 80 µL 100 µL 120 µL

Test parameters

NC1 : 0.085 NC2 : 0.119 PC : 2.395


NC1 : 0.086 NC2 : 0.084 PC : 2.463


NC1 : 0.080 NC2 : 0.081 PC : 2.481




Lot 8A


Test parameters

NC1 : 0.097 NC2 : 0.117 PC : 3.063


NC1 : 0.091 NC2 : 0.091 PC : 3.099


NC1 : 0.090 NC2 : 0.116 PC : 2.874




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Lot 12A


Test parameters

NC1 : 0.101 NC2 : 0.114 PC : 2.539


NC1 : 0.089 NC2 : 0.103 PC : 3.079


NC1 : 0.084 NC2 : 0.083 PC : 2.342




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Table 9 - Ruggedness Study - Variation of the conjugate volume

Lot 2A

Conjugate volume 80 µL 100 µL 120 µL

Test parameters

NC1 : 0.076 NC2 : 0.083 PC : 2.165


NC1 : 0.079 NC2 : 0.080 PC : 2.133


NC1 : 0.081 NC2 : 0.085 PC : 2.306




Lot 8A


Test parameters

NC1 : 0.077 NC2 : 0.075 PC : 2.190


NC1 : 0.076 NC2 : 0.077 PC : 2.588


NC1 : 0.087 NC2 : 0.138 PC : 2.550




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Lot 12A


Test parameters

NC1 : 0.083 NC2 : 0.104 PC : 2.506


NC1 : 0.078 NC2 : 0.079 PC : 2.660


NC1 : 0.092 NC2 : 0.090 PC : 2.602




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35

Appendix 1: Flow diagram showing the ISO 6579:2002 method and the Transia Plate Salmonella Gold test for the detection of Salmonella spp.

Salmonella cultured in M broth or Nutrient broth incubated overnight at 37°C

(or contamination by a freeze-dried bacterial culture)

Food matrix inoculation at 1-10 cfu/25g

Storage of food portions at 2-8°C or 18-25°C for 72h

MPN analysis : Weigh of 3 x 100g, 3 x 10g, 3 x 1g and 3 x 0.1g from the inoculated and uninoculated food portions; pre-enrichment in BPW at 37 ± 1°C for 16-20h.

Samples : Weigh of 5 x 25g uninoculated samples and 20 x 25g inoculated samples; pre-enrichment with 225ml of BPW at 37 ± 1°C for 16-20 h.

0.1ml BPW into 10ml RVS. Incubation at 41.5 ± 1 °C for 18-24 h

1ml BPW into 10ml MKTTn. Incubation at 37 ± 1 °C for 21-27 h.

0.1ml BPW into 10ml RVS. Incubation at 41.5 ± 0.5 °C for 18-24 h .

1ml BPW into 10ml MKTTn. Incubation at 37 ± 1°C for 21-27 h.

Streaking onto XLD and BGA plates. Incubation at 37 ± 1 °C for 21-27 h

Heat-treatment of 1 or 2ml RVS at 100°C for 20 minutes in a boiling water. Cool to room temperature

Transia Plate Salmonella Gold Confirmation of presumptive positive colonies by biochemical (API 20E or Microbact 24E) and serological tests (poly O and poly H), on upto 5 colonies per sample

ISO method TPSG

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Appendix 2: Comparison of TPSG and ISO results from uninoculated and inoculated cooked chicken.

Transia Plate Salmonella Gold

ISO Culture method

Sample code

Contamination level / strain

Optical density (OD)

Results Confirmation Presumptive positive colonies

Confirmation

8 0,129 - NT - NT 15 0,113 - NT - NT 27 0,120 - NT - NT 32

0 cfu/25g 0,126 - NT - NT

36 0,124 - NT - NT Total 0/5 0/5 0/5

6 0,783 + + + + 7 1,031 + + + +

12 0,393 + + + + 13 0,997 + + + + 14 0,774 + + + + 16 1,230 + + + + 18 0,854 + + + + 20 0,805 + + + + 21 0,758 + + + + 22 0,841 + + + + 23 0,507 + + + + 25 0,117 - NT - NT 26 1,135 + + + + 28 0,122 - NT - NT 31 0,718 + + + + 35 0,828 + + + + 39 0,803 + + + + 40 0,839 + + + + 41

6 cfu/25g

Salmonella Kentucky

0,117 - NT - NT 42 0,115 - NT - NT

Total 16/20 16/20 16/20

NT = not tested + = positive - = negative Transia plate Salmonella Gold test parameters: OD values of kit controls: Positive control = 2.915 Negative control 1 = 0.085 Negative control 2 = 0.093 Positive threshold = (0.085 +0.093)/2 + 0.11 = 0.199 Negative threshold = 0.199 x 0.9 = 0.180 OD value for a positive sample ≥ 0.199 OD value for a negative sample < 0.180

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Appendix 3: Comparison of TPSG and ISO results from uninoculated and inoculated cantaloupe.


ISO Culture method

Sample code



Results Confirmation

Presumptive positive colonies

Confirmation

209 0,116 - NT - NT 217 0,111 - NT - NT 218 0,097 - NT - NT 236

0 cfu/25g 0,104 - NT - NT

246 0,100 - NT - NT Total 0/5 0/5 0/5

206 0,398 + + + + 207 0,449 + + + + 212 0,490 + + + + 215 0,435 + + + + 219 0,471 + + + + 221 0,400 + + + + 222 0,416 + + + + 224 0,440 + + + + 226 0,495 + + + + 230 0,421 + + + + 231 0,461 + + + + 232 0,495 + + + + 234 0,538 + + + + 238 0,413 + + + + 242 0,395 + + + + 243 0,458 + + + + 244 0,396 + + + + 247 0,106 - NT - NT 248

7 cfu/25g

Salmonella Poona

0,403 + + + + 250 0,425 + + + +

Total 19/20 19/20 19/20


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Appendix 4: Comparison of TPSG and ISO results from uninoculated and inoculated sausages.


ISO Culture method

Sample code




Confirmation

310 0,093 - NT - NT 316 0,103 - NT - NT 323 0,117 - NT d - 332

0 cfu/25g 0,099 - NT - NT

341 0,100 - NT - NT Total 0/5 0/5 0/5

306 1,287 + + + + 309 1,436 + + + + 312 0,111 - NT - NT 313 1,518 + + + + 315 1,603 + + + + 317 1,297 + + + + 320 1,184 + + + + 324 1,527 + + + + 325 1,496 + + + + 328 1,708 + + + + 329 1,632 + + + + 331 1,854 + + + + 334 0,095 - NT - NT 337 1,685 + + + + 340 1,662 + + + + 343 1,643 + + + + 346 1,567 + + + + 347 1,711 + + + + 349

4 cfu/25g

Salmonella Bredeney

1,799 + + + + 350 1,687 + + + +

Total 18/20 18/20 18/20

NT = not tested + = positive - = negative d = doubtful presumptive positive colonies Transia plate Salmonella Gold test parameters: OD values of kit controls: Positive control = 3.079 Negative control 1 = 0.083 Negative control 2 = 0.088 Positive threshold = (0.083 +0.088)/2 + 0.11 = 0.196 Negative threshold = 0.1955 x 0.9 = 0.176 OD value for a positive sample ≥ 0.196 OD value for a negative sample < 0.176

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Appendix 5: Comparison of TPSG and ISO results from uninoculated and inoculated raw shrimps.


ISO Culture method

Sample code




Confirmation

409 0,109 - NT - NT 419 0,108 - NT - NT 428 0,111 - NT - NT 439

0 cfu/25g 0,098 - NT - NT

446 0,097 - NT - NT Total 0/5 0/5 0/5

406 2,085 + + + + 407 0,104 - NT - NT 410 2,169 + + + + 411 0,109 - NT - NT 413 0,113 - NT - NT 415 0,113 - NT - NT 418 2,475 + + + + 421 2,310 + + + + 422 2,318 + + + + 423 2,084 + + + + 427 2,041 + + + + 430 2,178 + + + + 432 0,097 - NT - NT 435 1,853 + + + + 437 1,906 + + + + 441 1,828 + + + + 442 1,620 + + + + 445 2,250 + + + + 449

6 cfu/25g

Salmonella Montevideo

2,272 + + + + 450 2,259 + + + +

Total 15/20 15/20 15/20


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Appendix 6: Comparison of TPSG and ISO results from uninoculated and inoculated yogurt.


ISO Culture method

Sample code




Confirmation

508 0,097 - NT - NT 522 0,104 - NT - NT 532 0,094 - NT - NT 541

0 cfu/25g 0,095 - NT - NT

542 0,100 - NT - NT Total 0/5 0/5 0/5

506 1,357 + + + + 507 0,099 - NT - NT 510 1,334 + + + + 513 0,104 - NT - NT 515 1,414 + + + + 517 1,398 + + + + 520 1,386 + + + + 523 1,364 + + + + 527 1,414 + + + + 528 0,093 - NT - NT 531 0,098 - NT - NT 533 0,090 - NT - NT 535 1,381 + + + + 538 1,424 + + + + 539 1,467 + + + + 540 0,095 - NT - NT 543 1,345 + + + + 546 1,344 + + + + 548

1 cfu/25g

Salmonella Typhimurium

1,265 + + + + 550 1,296 + + + +

Total 14/20 14/20 14/20


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Appendix 7: Comparison of TPSG and ISO results from uninoculated and inoculated mayonnaise.


ISO Culture method

Sample code



Results Confimation Presumptive positive colonies

Confirmation

1811 0,120 - NT - NT 1817 0,130 - NT - NT 1823 0,129 - NT - NT 1828

0 cfu/25g 0,128 - NT - NT

1835 0,119 - NT - NT Total 0/5 0/5 0/5

1806 0,117 - NT - NT 1807 0,130 - NT - NT 1812 0,119 - NT - NT 1813 1,824 + + + + 1814 0,143 - NT - NT 1818 1,724 + + + + 1819 0,129 - NT - NT 1822 0,125 - NT - NT 1825 0,136 - NT - NT 1827 0,122 - NT - NT 1830 0,148 - NT - NT 1832 1,809 + + + + 1833 0,128 - NT - NT 1836 0,124 - NT - NT 1839 0,131 - NT - NT 1840 0,144 - NT - NT 1843 1,599 + + + + 1845 1,838 + + + + 1847

1 cfu/25g

Salmonella Heidelberg

1,833 + + + + 1849 0,142 - NT - NT

Total 6/20 6/20 6/20


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Appendix 8: Comparison of TPSG and ISO results from uninoculated and inoculated shell eggs.


ISO Culture method

Sample code




Confirmation

709 0,114 - NT - NT 710 0,137 - NT - NT 726 0,118 - NT - NT 733

0 cfu/25g 0,125 - NT - NT

749 0,100 - NT - NT Total 0/5 0/5 0/5

706 2,591 + + + + 707 1,719 + + + + 708 0,105 - NT - NT 711 2,523 + + + + 714 0,108 - NT - NT 715 2,862 + + + + 719 2,249 + + + + 720 0,106 - NT - NT 721 2,299 + + + + 723 2,037 + + + + 728 2,918 + + + + 729 2,995 + + + + 730 0,112 - NT - NT 731 2,930 + + + + 735 2,034 + + + + 736 0,109 - NT - NT 740 2,565 + + + + 742 2,662 + + + + 746

1 cfu/25g

Salmonella Enteritidis

0,107 - NT d - 747 0,110 - NT - NT

Total 13/20 13/20 13/20


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Appendix 9: Comparison of TPSG and ISO results from uninoculated and inoculated frozen berries.


ISO Culture method

Sample code




Confirmation

2909 0,106 - NT - NT 2918 0,098 - NT - NT 2926 0,103 - NT - NT 2942

0 cfu/25g 0,113 - NT - NT

2959 0,112 - NT - NT Total 0/5 0/5 0/5

2906 1,065 + + + + 2910 0,101 - NT - NT 2912 1,151 + + + + 2913 0,101 - NT - NT 2919 1,086 + + + + 2920 0,113 - NT - NT 2924 1,175 + + + + 2927 1,095 + + + + 2929 1,134 + + + + 2930 0,105 - NT - NT 2934 0,108 - NT - NT 2935 0,106 - NT - NT 2938 1,136 + + + + 2939 1,166 + + + + 2940 1,127 + + + + 2947 1,282 + + + + 2948 1,118 + + + + 2951 1,142 + + + + 2952

4 cfu/25g

Salmonella Newport

0,114 - NT - NT 2958 1,207 + + + +

Total 13/20 13/20 13/20


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44

Appendix 10: Comparison of TPSG and ISO results from uninoculated and inoculated bean sprouts.


ISO Culture method

Sample code




Confirmation

908 0,098 - NT - NT 915 0,098 - NT - NT 916 0,115 - NT - NT 929

0 cfu/25g

0,104 - NT - NT 942 0,116 - NT - NT

Total 0/5 0/5 0/5 906 2,030 + + + + 907 0,098 - NT - NT 909 1,671 + + + + 910 2,306 + + + + 911 2,282 + + + + 921 2,232 + + + + 922 1,627 + + + + 923 0,100 - NT d - 926 2,264 + + + + 928 0,117 - NT - NT 932 2,534 + + + + 933 2,402 + + + + 934 2,629 + + + + 935 2,308 + + + + 936 1,809 + + + + 943 1,456 + + + + 945 2,268 + + + + 947 2,375 + + + + 948

1 cfu/25g

Salmonella Infantis

2,417 + + + + 949 2,137 + + + +

Total 17/20 17/20 17/20


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Appendix 11: Comparison of TPSG and ISO results from uninoculated and inoculated raw ground beef.


ISO Culture method

Sample code




Confirmation

1009 0,118 - NT - NT 1020 0,106 - NT d - 1021 0,105 - NT - NT 1034

0 cfu/25g

0,107 - NT - NT 1041 0,105 - NT - NT

Total 0/5 0/5 0/5 1006 0,105 - NT - NT 1011 2,091 + + + + 1012 1,900 + + + + 1014 2,005 + + + + 1015 1,926 + + + + 1017 1,919 + + + + 1022 2,073 + + + + 1026 0,110 - NT d - 1027 1,599 + + + + 1028 1,885 + + + + 1029 2,079 + + + + 1030 2,075 + + + + 1032 2,137 + + + + 1035 0,098 - NT d - 1036 1,955 + + + + 1037 2,012 + + + + 1039 2,020 + + + + 1042 2,012 + + + + 1048

2 cfu/25g

Salmonella Anatum

2,045 + + + + 1050 2,104 + + + +

Total 17/20 17/20 17/20


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Appendix 12: Comparison of TPSG and ISO results from uninoculated and inoculated smoked fish.


ISO Culture method

Sample code




Confirmation

1111 0,115 - NT d - 1122 0,112 - - d - 1127 0,120 - NT - NT 1133

0 cfu/25g

0,125 - NT - NT 1147 0,142 - NT - NT

Total 0/5 0/5 0/5 1106 0,109 - NT - NT 1107 2,058 + + + + 1112 0,111 - NT - NT 1114 2,127 + + + + 1117 2,158 + + + + 1123 2,522 + + + + 1124 2,091 + + + + 1125 2,742 + + + + 1129 0,119 - NT - NT 1130 0,114 - NT - NT 1134 2,042 + + + + 1135 1,999 + + + + 1136 0,131 - NT d - 1137 2,032 + + + + 1138 0,114 - NT d - 1139 2,211 + + + + 1140 2,472 + + + + 1148 1,843 + + + + 1149

2 cfu/25g

Salmonella Virchow

1,756 + + + + 1150 2,438 + + + +

Total 14/20 14/20 14/20


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47

Appendix 13: Comparison of TPSG and ISO results from uninoculated and inoculated uncooked noodles.


ISO Culture method

Sample code




Confirmation

2808 0,107 - NT - NT 2811 0,123 - NT - NT 2814 0,115 - NT - NT 2815

0 cfu/25g

0,112 - NT - NT 2823 0,116 - NT - NT

Total 0/5 0/5 0/5 2806 0,962 + + + + 2807 0,907 + + + + 2809 0,793 + + + + 2810 0,859 + + + + 2812 0,886 + + + + 2813 0,546 + + + + 2816 0,830 + + + + 2817 0,774 + + + + 2818 0,799 + + + + 2819 0,925 + + + + 2820 0,812 + + + + 2821 0,790 + + + + 2822 0,785 + + + + 2824 0,976 + + + + 2825 0,702 + + + + 2826 0,903 + + + + 2827 0,119 - NT - NT 2828 0,767 + + + + 2829

1 cfu/25g

Salmonella IIIb 61:z:1.5

1,186 + + + + 2830 0,439 + + + +

Total 19/20 19/20 19/20


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48

Appendix 14: Comparison of TPSG and ISO results from uninoculated and inoculated milk chocolate.


ISO Culture method

Sample code




Confirmation

2313 0,128 - NT - NT 2318 0,112 - NT - NT 2324 0,127 - NT - NT 2335

0 cfu/25g

0,123 - NT - NT 2341 0,112 - NT - NT

Total 0/5 0/5 0/5 2306 2,523 + + + + 2307 2,693 + + + + 2308 2,760 + + + + 2314 2,684 + + + + 2316 2,695 + + + + 2319 2,558 + + + + 2320 2,641 + + + + 2325 2,690 + + + + 2326 2,792 + + + + 2327 2,767 + + + + 2329 0,120 - NT - NT 2332 2,671 + + + + 2333 0,142 - NT - NT 2336 2,620 + + + + 2337 2,303 + + + + 2342 2,826 + + + + 2345 1,896 + + + + 2347 1,941 + + + + 2349

2 cfu/25g

Salmonella Senftenberg

0,128 - NT - NT 2350 2,016 + + + +

Total 17/20 17/20 17/20


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49

Appendix 15: Comparison of TPSG and ISO results from uninoculated and inoculated black pepper.


ISO Culture method

Sample code




Confirmation

1408 0,121 - NT - NT 1418 0,121 - NT - NT 1429 0,123 - NT - NT 1443

0 cfu/25g

0,123 - NT - NT 1444 0,123 - NT - NT

Total 0/5 0/5 0/5 1406 0,117 - NT - NT 1407 0,121 - NT - NT 1410 0,129 - NT - NT 1411 2,683 + + + + 1414 2,927 + + + + 1415 0,118 - NT - NT 1421 0,116 - NT - NT 1422 2,615 + + + + 1423 2,846 + + + + 1427 2,864 + + + + 1432 2,639 + + + + 1434 3,001 + + + + 1436 2,700 + + + + 1438 2,890 + + + + 1440 3,107 + + + + 1441 3,038 + + + + 1446 2,812 + + + + 1447 2,514 + + + + 1449

10 cfu/25g

Salmonella Indiana

0,132 - NT - NT 1450 2,787 + + + +

Total 14/20 14/20 14/20


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50

Appendix 16: Comparison of TPSG and ISO results from uninoculated and inoculated cake mix.


ISO Culture method

Sample code




Confirmation

2419 0,143 - NT - NT 2424 0,142 - NT - NT 2432 0,145 - NT - NT 2440

0 cfu/25g

0,151 - NT - NT 2443 0,158 - NT - NT

Total 0/5 0/5 0/5 2406 2,066 + + + + 2407 2,316 + + + + 2408 0,986 + + + + 2409 2,018 + + + + 2412 1,918 + + + + 2415 2,272 + + + + 2420 2,302 + + + + 2421 0,154 - NT - NT 2425 2,271 + + + + 2429 1,983 + + + + 2430 1,855 + + + + 2434 2,359 + + + + 2435 2,055 + + + + 2437 2,145 + + + + 2441 2,626 + + + + 2442 2,375 + + + + 2445 2,158 + + + + 2446 2,431 + + + + 2447

6 cfu/25g

Salmonella Thompson

2,177 + + + + 2449 2,741 + + + +

Total 19/20 19/20 19/20


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51

Appendix 17: Comparison of TPSG and ISO results from uninoculated and inoculated infant formula.


ISO Culture method

Sample code




Confirmation

2212 0,111 - NT - NT 2216 0,119 - NT - NT 2224 0,135 - NT - NT 2227

0 cfu/25g

0,129 - NT - NT 2235 0,148 - NT - NT

Total 0/5 0/5 0/5 2207 0,837 + + + + 2208 0,963 + + + + 2209 0,939 + + + + 2220 0,960 + + + + 2221 0,801 + + + + 2222 0,909 + + + + 2223 0,124 - NT - NT 2225 0,134 - NT - NT 2226 0,126 - NT - NT 2228 0,908 + + + + 2229 0,923 + + + + 2230 0,133 - NT - NT 2238 0,971 + + + + 2239 0,142 - NT - NT 2240 0,920 + + + + 2244 0,105 - NT - NT 2246 0,129 - NT - NT 2248 0,132 - NT - NT 2250

1 cfu/25g

Salmonella Blockley

0,136 - NT - NT 2207 0,127 - NT - NT

Total 10/20 10/20 10/20


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52

Appendix 18: Comparison of TPSG and ISO results from uninoculated and inoculated dry pet food.


ISO Culture method

Sample code




Confirmation

2608 0,112 - NT - NT 2610 0,102 - NT - NT 2615 0,101 - NT - NT 2616

0 cfu/25g

0,096 - NT - NT 2629 0,106 - NT - NT

Total 0/5 0/5 0/5 2611 0,107 - NT - NT 2612 2,219 + + + + 2617 0,100 - NT - NT 2620 0,110 - NT - NT 2621 2,599 + + + + 2622 2,528 + + + + 2624 0,103 - NT - NT 2626 0,112 - NT - NT 2627 0,101 - NT - NT 2628 2,840 + + + + 2633 2,053 + + + + 2634 0,103 - NT d - 2635 2,426 + + + + 2636 2,252 + + + + 2638 2,272 + + + + 2640 2,471 + + + + 2642 2,439 + + + + 2644 0,115 - NT - NT 2645

2 cfu/25g

Salmonella Mbandaka

2,228 + + + + 2649 0,117 - NT - NT

Total 11/20 11/20 11/20


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53

Appendix 19: Comparison of TPSG and ISO results from uninoculated and inoculated raw milk.


ISO Culture method

Sample code




Confirmation

2708 0,126 - NT d - 2711 0,129 - NT d - 2729 0,141 - NT d - 2744

0 cfu/25g

0,122 - NT - NT 2747 0,133 - NT - NT

Total 0/5 0/5 0/5 2709 0,200 + / - + d - 2710 1,989 + + + + 2714 0,237 + + + + 2715 0,129 - NT d - 2716 0,141 - NT d - 2717 0,214 + / - + + + 2718 2,177 + + - NT 2719 0,130 - NT d - 2723 1,628 + + d - 2725 0,175 - NT - NT 2727 0,125 - NT d - 2731 0,137 - NT - NT 2732 0,129 - NT d - 2738 0,190 - NT + + 2739 0,142 - NT d - 2742 0,143 - NT - NT 2743 0,139 - NT d + 2745 0,158 - NT - NT 2746

1 cfu/25g

Salmonella Dublin

0,144 - NT d - 2749 0,143 - NT d -

Total 6/20 6/20 5/20

NT = not tested + = positive - = negative +/- = doubtful sample d = doubtful presumptive positive colonies Transia plate Salmonella Gold test parameters: OD values of kit controls: Positive control = 3.629 Negative control 1 = 0.097 Negative control 2 = 0.117 Positive threshold = (0.097 +0.117)/2 + 0.11 = 0.217 Negative threshold = 0.217 x 0.9 = 0.195 OD value for a positive sample ≥ 0.217 OD value for a negative sample < 0.195

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54

Appendix 20. Comparison of TPSG and ISO results from uninoculated raw ground turkey

Transia Plate Salmonella Gold ISO Culture Method Sample code Optical Density Results Presumptive Confirmed

27 0.112 - - NT

30 0.115 - - NT

33 0.104 - - NT

44 0.103 - - NT

48 0.107 - - NT

Total 0/5 0/5 0/5

NT = Not tested + = Positive - = Negative Transia Plate Salmonella Gold test parameters: Optical Density (OD) values of kit controls: Positive control = 2.672

Negative control 1 = 0.109 Negative control 2 = 0.094

Positive threshold = ((0.109+0.094)/2) + 0.11 = 0.2115 Negative threshold = 0.2115 x 0.9 = 0.190 OD value for a positive sample ≥0.2115 OD value for a negative sample <0.190

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Appendix 20 (continued). Comparison of TPSG and ISO results from raw ground turkey contaminated with low levels (6 cfu/25g) of Salmonella Enteritidis (CCFRA 1004)


26 1.607 + + +

28 1.671 + + +

29 0.606 + + +

31 0.124 - - NT

32 1.300 + + +

34 1.479 + + +

35 1.377 + + +

36 1.630 + + +

37 1.541 + + +

38 0.114 - - NT

39 1.492 + + +

40 1.628 + + +

41 1.509 + + +

42 0.104 - - NT

43 1.354 + + +

45 0.959 + + +

46 1.550 + + +

47 1.333 + + +

49 1.604 + + +

50 1.622 + + +

Total 17/20 17/20 17/20




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Appendix 21. Comparison of TPSG and ISO results from uninoculated Brie cheese


6 0.115 - - NT

8 0.108 - - NT

9 0.109 - - NT

14 0.109 - - NT

19 0.102 - - NT

Total 0/5 0/5 0/5




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Appendix 21 (continued). Comparison of TPSG and ISO results from Brie cheese contaminated with low levels (2.3 cfu/25g) of Salmonella Typhimurium (CCFRA 1009)


1 0.904 + + +

2 0.710 + + +

3 0.122 - - NT

4 0.831 + + +

5 0.771 + + +

7 0.117 - - NT

10 0.105 - - NT

11 0.849 + + +

12 0.714 + + +

13 0.107 - - NT

15 0.113 - - NT

16 0.107 - - NT

17 0.561 + + +

18 0.557 + + +

20 0.571 + + +

21 0.698 + + +

22 0.735 + + +

23 0.106 - - NT

24 0.106 - - NT

25 0.630 + + +

Total 12/20 12/20 12/20




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Appendix 22: Calculation of performance indicators after generalized categorization of test samples Calculations applied on all results obtained on samples contaminated at the low level Transia Plate Salmonella Gold

method result

Positive Negative Total Positive N11 = 284 N12 = 2 N1● = 286 ISO 6579:2002

method result Negative N21 = 3 N22 = 111 N2● = 114 Total N●1 = 287 N●2 = 113 Total = 400

Chi-square = (( | N12 – N21 | ) – 1 )2 / ( N12 + N21 ) = (( | 2 – 3 | - 1 )2 / ( 2 + 3 ) = ( 1 – 1 )2 / 5 = 0 Sensitivity rate = N11 / N1● = 284 / 286 = 99.3 % Specificity rate = N22 / N2● = 111 / 114 = 97.4 % False negative rate = N12 / N1● = 2 / 286 = 0.7 % False positive rate = N21 / N2● = 3 / 114 = 2.6 %

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