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AOAC Validation Report

RapidChek® for the Detection of Salmonella species in Raw Meat and Poultry.

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RapidChek® for the Detection of Salmonella species in Raw Meat and Poultry

Abstract Rapidchek Salmonella Lateral Flow Assay from Strategic Diagnostic Inc. has been validated for raw meat and poultry with two enrichment protocols, a proprietary 24hr enrichment procedure and a 48hr enrichment procedure in reference to the standard USDA/FSIS cultural method. Raw ground beef, pork, chicken and turkey, as well as chicken carcass rinsate were tested. The 24hr enrichment uses a proprietary Rapidchek Salmonella medium for 5hrs at 42oC, followed by a transfer to Tetrathionate broth (Hajna, TTh) for an additional 19 hours. The 48hr enrichment uses Buffered Peptone Water as pre-enrichment for 20-24 hrs as recommended by USDA/FSIS, followed by a transfer to TTh only for an additional 24hr. Both Rapidchek methods were compared to the USDA/FSIS method for each of the matrices evaluated. Meat samples were spiked with cells of various Salmonella strains at an inoculum level ranging from 1 – 10 cells per 25g sample. A total of 100 spiked samples per method (N=5x20x2=200) were tested, and 88, 88 and 89 positives were reported with the Rapidchek 24hr, 48hr and the reference method, respectively. Non-spiked samples from ground beef, pork, chicken and turkey (n=5x2x5=50) were reported as negative in Salmonella by all three methods. Two (2) of the chicken rinse water non-spiked samples as in the 48hr enrichment were reported by both Rapidchek and reference method and were confirmed positives, indicating a natural contamination. The overall method agreement for the 24hr and 48hr methods to the reference method were 97% and 99%, respectively. In addition, the Rapidchek Salmonella assay was evaluated with 114 Salmonella strains and 55 non-Salmonella bacteria strains common to these food types. The results showed that the assay detected all the Salmonella strains and none of the non-Salmonella strains, indicating a 100% sensitivity (inclusivity) and a 100% specificity (exclusivity). This study demonstrated that Rapidchek Salmonella assay with the recommended 24hr and 48hr enrichments performs as well as the USDA/FSIS method for the detection of Salmonella species in raw meat and poultry. Method Authors Jingkun Li, Orla Cloak, Meredith Sutzko, and George Teaney Strategic Diagnostics Inc., 128 Sandy Drive, Newark, Delaware 19713-1147 Submitting Company Strategic Diagnostics Inc., 111 Pencader Dr., Drive, Newark, Delaware 19702 Independent Laboratories Silliker Corporate Research Center, 160 Amory Drive, South Holland, IL 60473 Reviewers

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1.0 Scope of Method

1.1 Target organisms – Salmonella species

1.2 Matrices – Raw ground beef, raw ground pork, raw ground chicken, raw ground turkey and chicken carcass washes

1.3 Summary of validated performance claims – The LFD Test Method for

Salmonella species was evaluated and was shown to have 100% specificity (exclusivity) and 100% sensitivity (inclusivity) in this study. The accuracy of the LFD Test used with the Proprietary enrichment system for raw meat and poultry averaged 100%. Similarly, the accuracy of the LFD used with the reference enrichment system (USDA/FSIS Microbiology Laboratory Guidebook , 3rd Edition, Rev #1, 9/6/99, Chapter 4) averaged 100%. The method agreement between the LFD Test methods and the USDA/FSIS for the detection of Salmonella in raw ground meat and poultry averaged 98%.

2.0 Definitions

2.1 Sensitivity – Rate of sensitivity = (No. of LFD Test Positives)/(No. Tested) × 100

2.2 Specificity – Rate of specificity = (No. of LFD Test Negative)/(No. Tested) × 100

2.3 Accuracy – (No. of Confirmed Positives/Total No. Positives Tested) × 100

2.4 Method Agreement. – Method Agreement = [1- (No. of Correct LFD Test – No. of Correct Reference Method / Total No. Method Samples)] × 100.

3.0 Principle

3.1 The LFD Test for Salmonella species can be used in combination with the proprietary enrichment system for a rapid 24 hour test or with currently recommended USDA media for a 48 hour test. After suitable enrichment, the sample broth is dispensed into the sample port of the LFD. The sample flows through a zone containing antibody coated colloidal gold reagents specific to Salmonella species. If antigens are present in the sample, they will bind to the antibody-conjugates to form an antigen/antibody complex. As this complex migrates through the nitrocellulose matrix, it passes a zone of anti-Salmonella antibody. If antigen is present, the complex is captured in this zone and is visualized by the formation of a red line. A second zone on the membrane is designed to capture any antibody-gold complex not bound in the first zone. As a result, when Salmonella antigen is present, the formation of 2 red lines is observed, whereas when no Salmonella is present, only 1 line forms.

4.0 General Information

4.1 Salmonella is an important human pathogen, which has been implicated as a major cause of foodborne illness worldwide. Each year, this organism is responsible for approximately 1.4 million cases of illness in the United States,

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95% of which are contracted through foodborne transmission, 600 of these typically result in death (1). Clinical symptoms of Salmonella vary, ranging from mild gastroenteritis to more serious conditions including septicemia and death (2). Raw meat and poultry are recognized as important vehicles of transmission of Salmonella (3, 4). Several control measures have been implemented along the production lines in meat and poultry abattoirs to reduce the levels of Salmonella in these matrices; however, microbiological testing at production levels retains a key role in preventing foodborne salmonellosis. Various methods have been developed to allow reduction in time required for the detection of the pathogen (5), as standard cultural methods for the detection of Salmonella in foods is time consuming and laborious requiring 4-5 days for a confirmed result (6, 7). This type of cultural confirmation is not only unsuitable for the routine monitoring of large volumes of samples but also time to result is unfavorable for testing of perishable foods such as meat and poultry. A new method for the detection of Salmonella in foods in a minimum of 24 h has been developed by Strategic Diagnostics Inc. This method utilizes specific antibodies in a lateral flow format, which are directed against surface antigens expressed in a large variety of Salmonella species. The objective of the present study was to determine to sensitivity, specificity and applicability of this novel system in raw meat and poultry matrices.

5.0 Test Kit Information

5.1 Kit Name – RapidChek Salmonella test kit, includes 50 lateral flow devices, with the option of a proprietary Rapid✔Media for Salmonenlla enrichment media which is included in the rapid 24 h test.

5.2 Catalog Number – 7000166

5.3 Ordering Information – Strategic Diagnostics Inc., 111 Pencader Drive, Newark,

DE 19702; Phone: (800) 544-8881 or www.sidx.com

6.0 Media and Reagents

6.1 RapidChek Salmonella Lateral Flow assay

6.2 Enrichment

6.2.1 RapidChek Media for Salmonella – (Strategic Diagnostics Incorporated, Newark, DE). Dissolve 27g in one liter of sterile distilled or deionized water prewarmed to 42oC. Use immediately for best results, media should be utilized within 3 hours.

6.2.2 Buffered Peptone Water (BPW) - (Difco Becton Dickinson). Dissolve

20g of powder media in one liter of sterile distilled or deionized water. Autoclave for 15 minutes at 121oC. Cool to room temperature.

6.2.3 TT (hajna) – add 91.5g of powder media to 1L of deinonized water, bring

to the boil and then cool to below 50°C, add 40 ml/l of iodine solution, dispense 10 ml into sterile test tubes.

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6.2.4 Rappaport Vassilidis (RV) – add 26.6g powder media to 1 L of deionized water and heat gently to dissolve media and dispense 10 ml into sterile tubes, autoclave 116°C for 15 mins.

6.3 Diagnostic reagents - Culture and biochemical reagents necessary for traditional confirmation of presumptive positives. See “Chapter 4 entitled Isolation and identification of Salmonella from meat, poultry and egg products (revision #1; 1/10/01) and Chapter 4A addendum entitled FSIS procedure for the use of Salmonella rapid screening immunoassay kits (released April 19th, 2001)” in USDA/FSIS Microbiology Laboratory Guidebook , 3rd Edition, Rev #1, 9/6/99. Web page: http://www.fsis.usda.gov/ophs/microlab/mlgbook.html

6.4 Disposable 200 µl pipette tips

6.5 Filtered stomacher bags

6.6 Inoculating needles and loops

6.7 Vortex mixer

6.8 Brillant green sulfa agar (BGS)

6.9 Xylose Lysine Tergitol 4 agar (XLT4)

6.10 Triple Iron Sugar Agar (TSI)

6.11 Lysine Iron Agar (LIA)

6.12 BioMerieux API20E kits for identification of Enterobacteriacae

6.13 Oxoid, Salmonella Latex Test

7.0 Apparatus

7.1 RapidChek Salmonella Lateral Flow devices( Figure 1).

7.2 Micropipet – Capable of accurately dispensing 150 µl. 7.3 Stationary incubator – Capable of holding temperature at 42oC for 20-24 hours. 7.4 Stationary incubator – Capable of holding temperature at 37oC for 20-24 hours. 7.5 Stomacher – Seward 400 Circulator (Seward, London, England) - for thorough

mixing of food samples in enrichment broth. 7.6 Top loading balance – To weigh 25 g ( + 0.1g) test samples.

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8.0 Standard Reference Material

8.1 Chapter 4 entitled Isolation and identification of Salmonella from meat, poultry

and egg products (revision #1; 1/10/01))” in USDA/FSIS Microbiology Laboratory Guidebook , 3rd Edition, Rev #1, 9/6/99. Web page: http://www.fsis.usda.gov/ophs/microlab/mlgbook.html

9.0 Safety Precautions

9.1 Live bacteria may be infectious and pose a significant health hazard. All bacterial cultures should be handled with caution and biohazard waste should be disposed of appropriately.

10.0 Sample Preparation

10.1 Raw ground beef, raw ground pork, raw ground turkey, raw chicken

10.1.1 Aseptically weigh a representative 25 g sample and add to filtered stomacher bag.

10.1.2 Add 225 ml of either BPW or RapidChek Media for Salmonella pre-

warmed to 42 oC.

10.1.3 Stomach samples for approximately 45 seconds.

10.1.4 Incubate BPW samples at 35oC for 20-24 h in a stationary incubator for the USDA reference method. Incubate Proprietary Media samples at 42oC for 5 h in a stationary incubator.

10.1.5 After 5 h, transfer 1 ml of the enriched RapidChek Media to 10 ml of

TT(hajna) broth and incubate at 42°C for 19 h.

10.1.6 After incubation, enriched RapidChek Media can be tested on LFD.

10.1.7 After 24 h, transfer 0.1 ml of the enriched BPW to 10 ml of RV and another 0.5 ml to 10 ml of TT (hajna) and incubate both enrichments at 42°C +/- 0.2°C for 24 h +/- 2 h.

10.1.8 After incubation, vortex both RV and TT (hajna) enrichments, test the TT

(hajna) enrichment only on the LFD. Both RV and TT (hajna) enrichments are simultaneously streaked to XLT4 and BGS plates according to the FSIS/USDA procedure.

10.1.9 All positive analyses are confirmed according to the FSIS/USDA

procedure.

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11.0 Set Up and Operation

11.1 Remove the required number of test cassettes from the foil pouch and place on a flat surface.

11.2 Label each cassette with the appropriate sample identification. 11.3 Take one pipette from the bag. Squeeze and hold the bubble on the top of the

pipette and place in the sample enrichment (samples enriched either in the Proprietary Media –TT (hajna) or BPW-TT(hajna).

11.4 Release the bulb and the required volume of solution (150uL) will rise in the

barrel of the pipette. 11.5 Hold the pipette over the sample reservoir on the cassette and add the entire

sample volume of the pipette barrel by squeezing the bulb. Discard the used pipette.

11.6 Let the cassette develop for 10 minutes.

12.0 Interpretation and Test Result Report

12.1 After a 10 minute time period, the appearance of one red line (control line) on the LFD Test indicates a negative result. The appearance of two red lines ( the control line and the test line) on the LFD Test indicates a positive result (Figure 1).

13.0 Independent Validation Studies

13.1 Validation studies were conducted by Silliker Laboratories in South Holland, Illnois, under the direction of the AOAC Research Institute.

13.2 Method Comparison Study - Raw ground turkey, was analyzed by the LFD Test

to determine the agreement of the kit performance relative to the current reference method for this food matrix. Replicate samples at two inoculum levels (zero and low) were examined for each matrix. Refer to Table 4 for a summary of the independent validation study results.

13.2.1 Methodology

13.2.1.1 Raw ground turkey

S. heidelberg (ATCC # 8326) was grown in 10 ml TSB broth at 37°C for 18 h. One thousand grams of raw ground turkey was inoculated with a low inoculum (1-10 cfu/25g) and homogenized by shaking and mixing manually in a large stomacher bag for 5 minutes. Samples were stressed, to mimic real life environmental conditions, by storing the batch of meat at 4oC for 48 h. An MPN (most probable number) was set up to determine cell levels after the stress period. Inoculated

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meat was divided into two sets of twenty 25 g samples. One set was stomached with 225 ml of the Proprietary Test Media for 45 seconds and incubated for 5 h at 42°C and then 1 ml of the enriched culture was transferred to 10 ml of TT (hajna) and incubated an additional 19 h at 42°C. Samples were tested on the lateral flow device as previously described.

A second set of twenty 25 g samples of inoculated turkey were enriched in 225 ml of BPW, which was incubated at 35°C +/- 2°C for 20-24 h and was processed according to the USDA/FSIS reference protocol for Salmonella, Chapter 4 (6).

All positive analysis were confirmed using the standard cultural and biochemical tests recommended by FSIS/USDA. In brief, this involves streaking samples from RV and TT (hajna) enrichments onto XLT4 and BGS. Typical Salmonella colonies which are well isolated on XLT4 and BGS plates are picked and screened by latex agglutination, and on TSI and LIA slants. Negative plates are reincubated for an additional 24 h and reexamined the following day. Further biochemical tests in the form of API20E were performed(6).

13.2.2 Results: 13.2.2.1 In the External laboratory study all of the non-inoculated

samples reported negative results (Table 4). Thirteen (13) of 20 inoculated samples were reported and confirmed positive using the 24hr method. Fifteen (15) of 20 samples were reported and confirmed positive by both the 48hr Rapidchek method as well as the USDA method.

14.0 Internal Validation Studies

14.1 Inclusivity Study

14.1.1 One hundred and fourteen (114) Salmonella strains (Table 1) were analyzed to determine the sensitivity of the LFD Test.

14.1.2 Methodology

14.1.2.1 One hundred and fourteen (114) Salmonella strains

were inoculated into individual 10 ml aliquots of the M-Broth and incubated at 37oC for 24 h. After enrichment, samples were tested on the LFD Test as described previously.

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14.1.3 Results

14.1.3.1 Results from the Inclusivity study are summarized in Table 3. As can be seen, the LFD Test was found to have a sensitivity of 100% for detecting specific strains of Salmonella from a variety of sources including clinical, environmental and food isolates.

14.2 Exclusivity Study

14.2.1 Fifty five (55) non-Salmonella strains (Table 3) were tested to determine

the specificity of the kit.

14.2.2 Methodology

14.2.2.1 Strains were grown at 37 oC for 24 h in 10 ml of TSB broth. After incubation, samples were evaluated using the LFD Test as described previously.

14.2.3 Results

14.2.3.1 Exclusivity results are summarized in Table 3. Of the 55 non-

Salmonella strains tested, all 55 were correctly identified as negative by the LFD Test, resulting in a specificity of 100%.

14.3 Method Comparison Study

14.3.1 Raw ground beef , raw ground pork, raw ground chicken and chicken

carcass washes were analyzed by the LFD Test to determine the agreement of the kit performance relative to the current reference method for each food matrix examined. Replicate samples at two inoculum levels (zero and low) were examined for each matrix.

14.3.2 Methodology

14.3.2.1 Raw ground beef

S. hadar (ATCC # 61777) was grown in 10 ml TSB broth for at 37°C for 18 h. One thousand grams of raw ground beef was inoculated with a low inoculum (1-10 cfu/25g) and homogenized by shaking in a large stomacher bag for 5 minutes. Samples were stressed, to mimic real life environmental conditions, by storing the inoculated batch of meat at 4oC for 48 h. An MPN (most probable number) was set up to determine cell levels after the stress period. Inoculated meat was divided into two sets of twenty 25 g samples. One set was stomached with 225 ml of the Proprietary Test Media for 45 seconds and incubated for 5 h at 42°C and

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then 1 ml of the enriched culture was transferred to 10 ml of TT (hajna) and incubated an additional 19 h at 42°C. Samples were tested on the lateral flow device as previously described.

A second set of twenty 25 g samples of inoculated meat were enriched in 225 ml of BPW, which was incubated at 35C for 20-24 h and was processed according to the USDA/FSIS reference protocol for Salmonella, Chapter 4 (6).

All positive analysis were confirmed using the standard cultural and biochemical tests recommended by FSIS/USDA as previously described.

14.3.2.2 Raw Ground Pork

S. agona (ATCC # 51957) was grown in 10 ml TSB broth for at 37°C for 18 h. One thousand grams of raw ground pork was inoculated with a low inoculum (1-10 cfu/25g) and homogenized by shaking in a large stomacher bag for 5 minutes. Samples were stressed, to mimic real life environmental conditions, by storing the batch of meat at 4oC for 48 h. An MPN (most probable number) was set up to determine cell levels after the stress period. Inoculated meat was divided into two sets of twenty 25 g samples. One set was stomached with 225 ml of the Proprietary Test Media for 45 seconds and incubated for 5 h at 42°C and then 1 ml of the enriched culture was transferred to 10 ml of TT (hajna) and incubated an additional 19 h at 42°C. Samples were tested on the lateral flow device as previously described. A second set of twenty 25 g samples of inoculated meat were enriched in 225 ml of BPW, which was incubated at 35C for 20-24 h. and was processed according to the USDA/FSIS reference protocol for Salmonella, Chapter 4 (6). All positive analysis were confirmed using the standard cultural and biochemical tests recommended by FSIS/USDA as previously described.

14.3.2.3 Raw Ground chicken

S. kentucky (ARS 25) was grown in 10 ml TSB broth for at 37°C for 18 h. One thousand grams of raw ground chicken was inoculated with a low inoculum (1-10 cfu/25g) and homogenized by shaking in a large stomacher bag for 5 minutes. Samples were stressed, to mimic real life environmental conditions, by storing the

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batch of ground chicken at 4oC for 48 h. An MPN (most probable number) was set up to determine cell levels after the stress period. Inoculated meat was divided into two sets of twenty 25 g samples. One set was stomached with 225 ml of the Proprietary Test Media for 45 seconds and incubated for 5 h at 42°C and then 1 ml of the enriched culture was transferred to 10 ml of TT (hajna) and incubated an additional 19 h at 42°C. Samples were tested on the lateral flow device as previously described. A second set of twenty 25 g samples of inoculated chicken were enriched in 225 ml of BPW, which was incubated at 35C for 20-24 h. and was processed according to the USDA/FSIS reference protocol for Salmonella, Chapter 4 (6). All positive analysis were confirmed using the standard cultural and biochemical tests recommended by FSIS/USDA as previously described.

14.3.2.4 Chicken Carcass Washes

S. enteriditis (ATCC # 13076) was grown in 10 ml TSB broth for at 37°C for 18 h. Forty (40) chicken pieces were individually inoculated on the surface with S. enteritidis at a low inoculum (1-10 cfu/sample) and placed in individual stomacher bags. Samples were stressed, to mimic real life environmental conditions, by storing at 4oC for 48 h. An MPN (most probable number) was set up to determine cell levels after the stress period. Each inoculated chicken piece, weighing ca. 100g were washed in 30 ml of BPW. The chicken pieces were rinsed with a rocking motion for approximately 30-60 seconds. The rinsate was then removed with a sterile pipette and transferred to a sterile stomacher bag with 225 ml of respective enrichment media. The remaining inoculated chicken piece was discarded. One set was stomached with 225 ml of the SDI Proprietary Test Media for 45 seconds and incubated for 5 h at 42°C and then 1 ml of the enriched culture was transferred to 10 ml of TT (hajna) and incubated an additional 19 h at 42°C. Samples were tested on the lateral flow device as previously described. A second set of twenty rinsate samples were enriched in 225 ml of BPW, which was incubated at 35°C for 20-24 h. and was processed according to the USDA/FSIS reference protocol for Salmonella, Chapter 4 (6). All

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positive analysis were confirmed using the standard cultural and biochemical tests recommended by FSIS/USDA.

14.3.3 Results

14.3.3.1 Results from the method comparison/repeatability study are summarized in Tables 5 - 6. In Ground Beef, the performance of the LFD Test with 24 h enrichment scheme and the 48 h enrichment scheme were found to compare favorably to the cultural method of detection with 100% accuracy and method agreement. The LFD Test-24 hour method recovered and positively identified 18 of the 20 inoculated samples, similarly the 48 h method correctly identified 18 of the 20 inoculated samples. The reference method also recovered 18 of the 20 inoculated samples, demonstrating a method agreement of 100%. Non-inoculated samples were found to be negative by both the 24 h and 48 h Rapidchek methods as well as the USDA/FSIS method.

14.3.3.2 A similar scenario was evident for raw ground chicken, with all

non-inoculated samples returning negative results by the 24 h and 48 h Rapidchek methods and USDA/FSIS method. The LFD Test-24 hour method recovered and positively identified 19 of the 20 inoculated samples, similarly the 48 h method correctly identified 19 of the 20 inoculated samples. The reference method also recovered 19 of the 20 inoculated samples. A 100% accuracy and method agreement was found with all the methods tested for this matrix.

14.3.3.3 For raw ground pork, the 24 h enrichment method recovered 19

of the 20 inoculated samples, all of which were confirmed culturally to be Salmonella. Both the 48 h Rapidchek method and the USDA/FSIS method were positively identified 18 of the 20 samples inoculated with Salmonella. All of the non-inoculated samples returned negative results for all 3 methods. For this matrix, a 100% accuracy and method agreement was evident with all methods examined.

14.3.3.4 In the chicken carcass wash study all of the non-inoculated

samples from the 24 h enrichment were negative. Two (2) of the non-inoculated samples from the 48 h enrichment returned positive results. The 24 h LFD method and the 48 h reference methods positively identified 19 of the 20 inoculated samples. The 48 h LFD method positively identified 18 of the 20 inoculated samples.

14.4 Ruggedness Studies

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14.4.1 The LFD Test is a very simple device to utilize. It is a compact open cassette which requires the edition of an aliquot of the enriched sample. Therefore, ruggedness testing for this study involved variation in temperature at which the device was tested, variations in times at which the strip was read, minor variations in sample preparation/enrichment times. Ruggedness testing additionally involved variations in sample volume added to the LFD Test, variability in time lots of the test strip used, and stability of test line after expiration of reading time has occurred.

14.4.2 Methodology

14.4.2.1 Temperature and Device Read Time Latitude Study - LFD

Tests were removed from their foil pouches and placed at 4°C, 20°C and 37°C to mimic potential temperature range at which the devices could be tested by the user. Both inoculated Salmonella meat samples and non inoculated samples were enriched with the 24 h enrichment and tested on the devices at 4°C, 20°C and 37°C. It is recommended in the package insert information for the LFD Test that 10 minutes is required prior to reading the strip and recording the result as negative for Salmonella. In addition to the assay run temperature evaluation, this study was designed to determine what effect a variation on reading the strip had on the end result of the assay. LFD Tests from the temperature study described above were read at 2 minute intervals over a period of 20 minutes.

14.4.2.2 Sample Volume Study - It is recommended in the package

insert for the LFD Test, that 150µl of the enriched sample be added to the port of the device. This study determined the effect of variations in the sample volume added to the device. Three (3) Salmonella positive (1-10 cfu/25g) and 3 negative (0 cfu/25g) proprietary media enriched ground pork samples were tested on the device. Volumes of 100µl, 125µl, 150µl, 200µl and 400µl of negative and positive samples were added to the device. LFD Tests were run and cassettes were opened after 10 minutes had expired and observations were made.

14.4.2.3 Lot to Lot Variability Study - Ground Pork samples were

inoculated with Salmonella at one level (4.4 cfu/sample, 20 sample) and enriched in the Proprietary Media for 24 h at 42°C. Five non-inoculated controls were run with this experiment. After incubation, samples were tested on 2 Production and 1 R&D lots of the LFD Tests to determine the lot to lot variability of the strips.

14.4.2.4 Stability Study - The stability of the LFD Test was examined

over a period of 46 days. LFD Tests were stored at 4°C, 25°C, 37°C and 45°C under desiccated conditions. Devices from an R&D Lot and a Manufacturing Lot were removed from their

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respective storage temperatures and tested with low and a high levels Salmonella calibrators. An negative controls were also examined. Samples were tested on the LFD Test at the following time points, day 0, 7, 14, 20, 28, 35.

14.4.2.5 Rapidchek Media Preparation Latitude – It is recommended in the package insert that the end user prepare the Rapidchek media by dissolving the appropriate mass of powdered media to the appropriate volume of autoclaved water. This preparation should be used within 4 hours. An alternative to this preparation method is to prepare and autoclave the media a day prior to the use. In this study ground turkey inoculated with S. heidelberg (ATCC # 8326) at 1.4 cfu/25g was enriched with Rapidchek salmonella media prepared either as described in the users guide (addition of media to autoclaved water) or by autoclaving media a day prior to the enrichment. Twenty inoculated and five non-inoculated samples were enriched using the 24hr procedure described above.

14.4.3 Results

14.4.3.1 The Salmonella LFD test was found to function properly

throughout the temperature and timing ranges described in section 14.4.2.1 (Table 7). Although in this study the correct result was observed as early as 2 minutes, it remains the recommendation of the manufacturer that a full ten minutes is allowed in order to properly identify negative samples.

14.4.3.2 The minimum volume of sample required to properly run the device is 125uL (Table 8). Sample volumes from 125 uL through 400uL all returned the same assay result, however the clarity and consistency of the resulting test line was slightly diminished at the 400uL sample volume (data not shown). Therefor the manufacture recommends the use of the 150uL disposable pipette supplied with the kit.

14.4.3.3 The Lot to Lot variability between two production lots and one R&D lot was evaluated (Table 9). No significant Lot to Lot differences were observed with the LFD product.

14.4.3.4 Results from the accelerated stability study report no decrease in performance from either LFD Lot at any of the temperature challenges over the course of the study (Table 10). These results indicate that the LFD test will have 1 year stability at room temperature and possibly longer duration under refrigerated conditions.

14.4.3.5 The use of autoclaved Rapidchek media does not appear to

effect the recovery of Salmonella from the sample (Table 11). Results from this study show 19 samples determined positive

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using autoclaved media, and 16 samples determined positive using non-autoclaved media.

15.0 Discussion

15.1 The 24hr and 48 hr Rapidchek methods have demonstrated excellent accuracy, sensitivity and specificity throughout these studies. Overall method agreement averaged 97% for the 24hr method and 99% for the 48hr method. The 2 methods have been shown to be capable of detecting very low levels of Salmonella in a variety of raw meat and poultry matrices. Sample matrix effects within the raw meat and poultry samples examined in these studies were not apparent. Assay robustness studies indicate that the assay will perform under a wide range of environmental conditions. The assay is stable for at least a year at room temperature and results are highly reproducible from lot to lot.

16.0 Conclusions

16.1 The use of the lateral flow test with both 24 h proprietary enrichment method and the 48 h enrichment scheme has demonstrated excellent accuracy, sensitivity and specificity throughout this study. The overall accuracy of these methods was 98%, with 100% sensitivity and specificity. There was excellent method agreement between the lateral flow system and the cultural confirmation. These studies demonstrates that low levels of Salmonella can be recovered and detected in a food matrix in a minimum of 24 h, this is a direct result of the enhanced resuscitation qualities of the proprietary media utilized in the initial stages of the enrichment procedure coupled with the low sensitivity and accuracy of the RapidChek Salmonella lateral flow device

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17.0 Reference

17.1 Mead, P.S., L., Slutsker, V. Dietz, F. McCraig, J.S. Bresee, C. Hapiro, P.M. Griffin, and R.V. Tauxe (1999) Food related illness and death in the United States. Emerging and Infectious Diseases, 5; 609-625.

17.2 D’Aoust, J.Y. (1997) Salmonella species: Chapter 8, pp 129-158; In: Food Microbiology, Fundamentals and Frontiers. Edited by Doyle, M.P., Beuchat, L. R. and Monteville, T.J. American Society of Microbiology, 1997.

17.3 Hargis, B.M., D.J., Caldwell, D.J., Brewer, R.L., Corrier, D.E., and J.R. Deloach. (1995) Evaluation of the chicken crop as a source of Salmonella contamination for broiler carcasses. Poultry Science, 74, 1548-1552.

17.4 Roels, T.H.P, P.A. Frazak, J.J. Kazmiercaxak, W.R., Mackenxie, M.E. Proctor, T.A. Kurzynski, and J.P. Davis. (1997) Incomplete sanitation of a meat grinder and ingestion of raw beef: contributing factors to a large outbreak of Salmonella typhimurium infection. Epidemiology and Infections, 119, 127-134.

17.5 Blackburn, C de W (1993) Review: Rapid and alternative methods for the detection of salmonellas in foods. Journal of Applied Bacteriology, 75, 199-214.

17.6 Rose, B.E. (1998) Isolation and identification of Salmonella from meat, poultry and egg products (chapter 4, revision #1; 1/10/01). In : USDA/FSIS Microbiology Laboratory Guidebook; website location: http://www.fsis.usda.gov/ophs/microlab/mlgbook.htm

17.7 Andrews, W.H. and T.S. Hammack (2001) Salmonella (chapter 5) In: US Food, Drug and Administration , Center for Food Safety and Applied Nutrition, Bacteriological Analytical Manual: website location: http://www.cfsan.fda.gov/~ebam/bam-5.html

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Figure 1: Image of the RapidChek lateral flow device for Salmonella.

Three RapidChek devices are shown, (a) one which has not be used; (b) one which has been run with a negative meat sample, displaying one red line indicative of a negative result; (c) one device which has been run with a positive meat sample, displaying two red lines indicative of a positive result.

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Table 1: Salmonella isolates source list – Inclusivity study Species and Sources Species and Source Species and Source

1 S. albany ATCCa 51960 39 S. heidelberg WVU 6F71 77 S. saintpaul FSIS 051

2 S. amherstiana DSMb 006 40 S. hvittingfoss DSM70 78 S. seftenburg 6F1

3 S. agona USDAc 41 S. infantis ARS 22 79 S. seftenburg 6F11

4 S. agona ATCC 51957 42 S. javiana ATCC 10721 80 S. seftenburg ARS 8

5 S. agona NFCd 1035: S-R2 43 S. jerusalem Tyson 25 81 S. stanley DSM 305

6 S. anatum ATCC 9270 44 S. kentucky DSM 7-82 82 S. thompson ATCC 8391

7 S. anatum 5F29 45 S. kentucky DSM 6-290 83 S. typhimurium ARS 3

8 S. anatum 5F28 46 S. kentucky DSM 7-19 84 S. typhimurium DSM 131-104

9 S. blockey DSM14 47 S. kentucky DSM 76-P 85 S. typhimurium DSM 128-NP

10 S. bredeney DSM 04 48 S. kentucky ATCC 9263 86 S. typhimurium DSM 126-193

11 S. brandenburg ARSe 20 49 S. kentucky DSM 7-147 87 S. typhimurium DSM 129-204C

12 S. brandenburg ARS 21 50 S. kentucky ARS 25 88 S. typhimurium DSM 124-193

13 S. brandenburg USDA-MFS 9190 51 S. kentucky ARS 26 89 S. typhimurium ATCC 23853

14 S. brandenburg DSM 15 52 S. livingstone M7 00-02940 90 S. typhimurium ATCC 15277

15 S. branderup DSM 16 53 S. london ATCC 8389 91 S. typhimurium ATCC 15421

16 S. clifton DSM 38 54 S. maarsen ATCC 15793 92 S. typhimurium ATCC 23555

17 S. choleraesusis Tyson 18 55 S. montevideo ARS 31 93 S. typhimruium ATCC 19585

18 S. cubana 12007-02 56 S. montevideo ARS 32 94 S. typhimurium ATCC 23657

19 S. dublin ATCC 15480 57 S. montevideo ARS 33 95 S. typhimurium ATCC 23595

20 S. derby DSM 58 S. meleagridis DSM 111 96 S. tyhimurium ATCC 23564

21 S. enteritidis ATCC 8391 59 S. muenchen ATCC 8388 97 S. typhimurium ATCC 7823

22 S. enteritidis M1 BGA 164/93 60 S. muenster WVU 5F30 98 S. typhimurium ATCC 23566

23 S. enteritidis T 22 61 S. muenster WVU 5F24 99 S. virginia DSM 145

24 S. enteritidis ARS 10 62 S. muenstar WVU 8F3 100 S. virchow DSM 143

25 S. enteritidis ARS 11 63 S. muenstar WVU 8F9 101 S. worthington 6F14

26 S. enteritidis ARS 12 – litter 64 S. muenstar WVU 5F22 102 S. worthington 6F51

27 S. enteritidis ATCC 13076 65 S. newbrunswick DSM 92 103 S. worthington ARS 146

28 S. frankfurt DSM 0417 66 S. newport ATCC 6962 104 S. witchita DSM 147

29 S. grumpiness DSM 328 67 S. newport ATCC H1275 105 S. typhimurium 14028

30 S. hadar ATCC 51956 68 S. oranienburg ATCC 9239 106 S. typhimurium 25241

31 S. hadar FSIS 044 69 S. paratyphi B ATCC 19940 107 S. mbandaka T23

32 S. heidelberg ATCC 8326 70 S. paratyphi B ATCC M4-00-02932 108 S. minnesota

33 S. heidelberg WVU 5F141 71 S. paratyphi C ATCC 1328 109 S. gallinarium

34 S. heidelberg WVU 5F105 72 S. poona DSM 109 110 S. panama T3

35 S. heidelberg WVU 5F130 73 S. poona DSM 338 111 S. reading 6967

36 S. heidelberg WVU 5F114 74 S. poona ARS 119 112 S. Thompson ARS 13

37 S. heidelberg WVU 5F140 75 S. poona ARS 121 113 S. Thompson ARS 14

38 S. heidelberg WVU 5F155 76 S. saintpaul ATCC 9712 114 S. Thompson ARS 15

19

Table 2: Non- Salmonella isolates

Species Source Species Source 1 Aeromonas hydropila 10 30 Proteus mirabilis 4630 2 Aeromonas hydropila 8 31 Proteus mirabilis 68 3 Bacillus cereus 11778 32 Proteus mirabilis 70 4 Escherichia coli O157:H7 35150 33 Proteus vulgaris 8427 5 Escherichia coli spp R7-32C4 34 Proteus vulgaris 19R7 6 Escherichia coli spp 96 C5 35 Enterobacter cloacae 13047 7 Escherichia coli spp 99 G1 36 Enterobacter cloacae 2 8 Escherichia coli spp 100D4 37 Enterobacter aerogenes 15038 9 Escherichia coli O86 ATCC 12701 38 Klebsiella pneumoniae 13883 10 Escherichia coli O111 33780 39 Klebsiella pneumoniae CK 11 Escherichia coli O91:NM 12795 40 Klebsiella pneumoniae 9 12 Escherichia coli O142 23980 41 Hafnia alvei - 13 Escherichia coli O55 51435 42 Proteus mirabilis 66Ta 14 Escherichia coli spp 29522 43 Listeria monocytogenes 4b 19115 15 Escherichia hermanii 33650 44 Listeria grayi - 16 Escherichia vulneris 33821 45 Serratia liquefaciens 27592 17 Escherichia blattae 33430 46 Serratia marcescens 264 18 Edwardsiella tarda 23663 47 Brochotrix thermosphacta 11509 19 Shigella spp 23354 48 Providencia stuartii - 20 Citrobacter freundi 8090 49 Yersinia enterocolitica - 21 Citrobacter freundi 3F9 50 V. parahaemolyticus - 22 Citrobacter freundi 3E4 51 R. equi - 23 Citrobacter freundi 5F8 52 L. mono 4b Scott A 24 Citrobacter sedlaki 51115 53 Vibrio 62A2 25 Citrobacter koseri 27026 54 C. freundii 68a1 26 Citrobacter braaki 51113 55 C. perfringens - 27 Citrobacter youngae 11102 28 Citrobacter farmeri 51112 29 Citrobacter werkmanii 51114

All strains for inclusivity and exclusivity were typed internally according to the USDA/FSIS confirmatory procedure (10).

20

Table 3: Inclusivity and Exclusivity Study

Organism Media type Total number of strains tested on RapidChek

Total positive on RapidChek

Sensitivity/ Specificity

(%)

Salmonella

species

M-Broth

114

114

100

Non Salmonella species

TSB 55 0 100

1

Table 4: External Method Comparison Study for the RapidChek Method with 24h Hour and 48 Hour RapidChek Method and the USDA/FSIS cultural method for raw turkey.

Sample Matrix

Method type Strain Inoculum Level

(cfu/25g)

MPN /25g

# Samples

Presumptive positives

ConfirmedPositives

% Accuracy

% method agreement

0 0 5 0 na 100 100 Rapidchek 24 h

1-10 2.3 20 13 13 100 90

0 0 5 0 Na 100 100 Rapidchek 48 h

1-10 2.3 20 15 15 100 100

0 0 5 0 Na 100 N/A

Ground Turkey

USDA/FSIS

Salmonella heidelberg

1-10 2.3 20 15 15 100 N/A

Background count for ground turkey:

N/A = not applicable

Table 5: The RapidChek Method with 24h Hour and 48 Hour RapidChek Method and the USDA/FSIS cultural method for beef and raw ground chicken.

Sample matrix

Method type Strain Inoculum Level

(cfu/25g)

MPN (cfu/25g)

# Samples

Presumptive Positives

ConfirmedPositives

% Accuracy

% method agreement

0 N/A 5 0 0 100 100 Rapidchek 24 h

2.8 4.3 20 18 18 100 100

0 N/A 5 0 0 100 100 Rapidchek 48 h

2.8 4.3 20 18 18 100 100

0 N/A 5 0 0 100 N/A

Ground Beef

USDA/FSIS

Salmonella hadar

2.8 4.3 20 18 18 100 N/A

Background count for ground beef: 5.6 x 10^4 cfu/g

0 N/A 5 0 0 100 100 Rapidchek 24 h

2.5 6.0 20 19 19 100 100

0 N/A 5 0 0 100 100 Rapidchek 48 h

2.5 6.0 20 19 19 100 100

0 N/A 5 0 0 100 N/A

Ground chicken

USDA/FSIS

Salmonella kentucky

2.5 6.0 20 19 19 100 N/A

Background count for ground chicken: 1.2 x 10^6 cfu/g

N/A = not applicable

Table 6: The RapidChek Method with 24h Hour and 48 Hour RapidChek Method and the USDA/FSIS cultural method for raw ground pork.

Sample matrix

Method type Strain Inoculum Level

(cfu/25g)

MPN Cfu/25g

# Samples

Presumptive Positives

ConfirmedPositives

% Accuracy

% method agreement

0 N/A 5 0 0 100 100 Rapidchek 24 h

2.5 9.0 20 19 19 100 95

0 N/A 5 0 0 100 100 Rapidchek 48 h

2.5 9.0 20 18 18 100 100

0 N/A 5 0 0 100 N/A

Ground Pork

USDA/FSIS

Salmonella agona

2.5 9.0 20 18 18 100 N/A

Background count for ground pork: 1.6 x 10^4 cfu/g

0 N/A 5 0 0 100 100 Rapidchek 24 h

4.4 2.3 20 19 19 100 100

0 N/A 5 2 2 100 100 Rapidchek 48 h

4.4 2.3 20 18 18 100 95

0 N/A 5 2 2 100 N/A

Chicken Carcass Wash

USDA/FSIS

Salmonella enteriditis

4.4 2.3 20 19 19 100 N/A

Background count for chicken carcass wash: 3.4 x 10^6 cfu/ml wash

N/A = not applicable

Table 7: Assay Temperature and Read Time Latitude Study

Inoculum Level of

Salmonella agona

Number of Samples Tested

Reading Time of Device

(minutes)

Number of RapidChek Positive Results at Various Temperatures/Total Tested

4 oC 20 oC 37 oC

0 cfu/25g (negative)

2.5 cfu/25g

5 5

2 4 6 8 10 12 14 16 18 20 2 4 6 8 10 12 14 16 18 20

0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5

5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5

0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5

5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5

0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5

5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5

Table 8: Assay Sample Volume Latitude Study Sample Volume (uL)

RapidChek Positive Results from Non-Inoculated Samples

RapidChek Positive Samples from Inoculated Samples

Accuracy (%)

100 125 150 200 400

0/3a 0/3 0/3 0/3 0/3

0/3a 3/3 3/3 3/3 3/3

0

100 100 100 100

a Not enough volume to run test

7

Table 9: Assay Lot to Lot Variability Latitude Study

Sample ID Inoculum Level (cfu/25g)

RapidChek Device Performance from 3 Different Device Lots

R&D Nb1871:79 MFG 2M1056 MFG 2M1067 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

2.5 2.5 0

2.5 2.5 0

2.5 2.5 2.5 2.5 2.5 2.5 0

2.5 2.5 2.5 2.5 0 0

2.5 0

2.5 2.5 2.5 2.5

+ + - + + - + + + + + + - + + + + - - + - + + + +

+ + - + + - + + + + + + - + + + + - - + - + + + +

+ + - + + - + + + + + + - + + + + - - + - + + + +

8

Table 10: Assay Accelerated Stability Study

Time (days)

Strip Lot Calibrator Level

RapidChek Device Results at Various Storage Temperatures

4 oC 25 oC 37 oC 45 oC 0

20

35

R&D

Nb:1871:79

MFG 2M1056

R&D

Nb:1871:79

MFG 2M1056

R&D

Nb:1871:79

MFG 2M1056

0

Low High

0

Low High

0

Low High

0

Low High

0

Low High

0

Low High

- + + - + + - + + - + + - + + - + +

- + + - + + - + + - + + - + + - + +

- + + - + + - + + - + + - + + - + +

- + + - + + - + + - + + - + + - + +

9

Table 11: RapidChek Media Preparation Latitude Study

Inoculation Level

(cfu/25g)

RapidChek Positives/Total Samples Tested

Non-Autoclaved Autoclaved

0

0/5

0/5

1.4

16/20

19/20