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[RapidChek SELECTTM SE AOAC Validation Report]

RapidChek SELECTTM

Salmonella Enteritidis Test System 1

for the Detection of Salmonella Enteritidis in Poultry House Drag Swabs, 2

Shell Egg Pools, and Chicken Carcass Rinsates 3

4

Abstract 5 6 The RapidChek SELECT

TM Salmonella Enteritidis Test System was validated for the 7

detection of Salmonella Enteritidis (SE) in poultry house drag swabs, shell egg pools, and 8 chicken carcass rinsates. The method utilizes RapidChek SELECT

TM Salmonella (AOAC 9

PTM License Number 080601) proprietary primary and secondary enrichment media. 10 Following enrichment, an immunochromatographic test strip is inserted into the tube 11 containing the secondary enrichment broth, developed for 10 min and interpreted. 12 Salmonella Enteritidis-inoculated samples (1 to 5 CFU SE / analytical unit) were tested by 13 the test method as well as the appropriate cultural reference method (FDA-BAM (drag 14 swabs and egg pools) or USDA-FSIS (chicken carcass rinsates)). A total of 80 samples 15 were tested by both methods in the study. Fifty-two (52) samples were positive by the 16 RapidChek SELECT

TM Salmonella Enteritidis method and 38 were found positive by the 17

respective reference method. The sensitivity of the method was 100% and the specificity 18 was 100%. The accuracy of the test method was 137%, indicating that the method was 19 more sensitive than the reference method. The RapidChek SELECT

TM Salmonella 20

Enteritidis method was tested with 82 Salmonella Group D1 strains including 63 Salmonella 21 Enteritidis strains as well as 32 non-Salmonella Group D1 strains representing 10 bacteria 22 genera. The test method detected all 82 Group D1 strains (100% sensitivity). None of the 23 non-Salmonella Group D1 or other genera of bacteria were detected, indicating a specificity 24 of 100%. The method was shown to be highly robust and stable under control and 25 accelerated stability conditions. 26 27 Method Author 28 29 Mark T. Muldoon, Ph.D., SDIX, 128 Sandy Drive, Newark, DE 19713 30 31 Submitting Company 32 33 SDIX, 111 Pencader Drive, Newark, DE 19702 34 35 Independent Laboratory 36 37 Q Laboratories, Inc.1400 Harrison Avenue, Cincinnati, OH 45214 38 39 Reviewers 40 41 Michael Brodsky, Ph.D., Brodsky Consultants, 73 Donnamore Crescent ,Thornhill, Ontario, 42 Canada L3T4K6 43 44 Edward Richter, Ph.D., Richter International, 1730 Atlas Street, Columbus, OH 43228 45 46


Thomas Hammack, Ph.D., AOAC General Referee 1 2 1. Scope of method 3 4

1.1 Target organisms – Salmonella Enteritidis and other Salmonella Group D1 bacteria 5 1.2 Matrices– Poultry house environmental drag swabs, egg pools, chicken carcass 6

rinsates 7 1.3 Summary of validated performance claims. — RapidChek SELECT

TM Salmonella 8

Enteritidis Test System was validated for the low level detection of Salmonella 9 Enteritidis (SE) (1-5 CFU/sample) in poultry house drag swabs, shell egg pools, and 10 chicken carcass rinsates. Method sensitivity was 100% and method specificity was 11 100%. Accuracy of the test method was 137%. 12

2. Definitions 13 14 2.1 Sensitivity .— Number of True Positives / (Number of True Positives + Number of 15

False Negatives) 16 2.2 Specificity .— Number of True Negatives / (Number of False Positives + Number of 17

True Negatives) 18 2.3 Accuracy.— Number of Confirmed Presumptive Positives for Test Method / 19

Number of Confirmed Positives for Reference Method 20 21

3. Principle 22 23

The RapidChek SELECTTM

Salmonella Enteritidis Test Kit is designed to detect 24 Salmonella Enteritidis (including other Group D1 serovars) in poultry house drag swabs, 25 shell egg pool samples and chicken carcass rinsate samples. The test kit permits the 26 presumptive detection and identification of the target pathogen in 40 or 48 hours, 27 dependent on sample type, when present at levels as low as 1-5 organisms per sample. 28 29 This immunoassay test uses a double antibody sandwich format in a lateral flow test 30 strip. It utilizes a murine monoclonal antibody specific for Salmonella Group D1 31 including Salmonella Enteritidis (SE). The antibody is sprayed and immobilized on the 32 surface of a nitrocellulose membrane comprising a “test line”. The same monoclonal 33 antibody is also labeled with colloidal gold and is contained within a reagent pad 34 upstream from the test line on the membrane. As the sample moves by capillary action 35 from the filter pad into the antibody–gold pad, the antibody–gold reagent specifically 36 binds to the target organism and moves with the liquid sample onto the test membrane. 37 The sample passes through the test line where the immobilized antibody captures the 38 antigen–antibody–gold complex, causing the formation of an antibody–antigen 39 “sandwich” and development of red color at the test line. Antibody–antigen sandwiches 40 are not formed in the absence of the Salmonella Group D1 including SE, resulting in no 41 red color development at the test line. Anti-mouse antibody immobilized at the control 42 line captures excess monoclonal antibody-gold reagent passing through the test line. The 43 presence of red color at the control line indicates that the strip has flowed correctly. 44 Therefore, the presence of only one line (control line) on the membrane indicates a 45 negative sample and the presence of two lines indicates a positive sample. 46


1 The immunomagnetic confirmation kit utilizes the same monoclonal antibody described 2 above attached to magnetic particles for the purification of SE and other Group D1 3 serovars from a complex enriched liquid media sample. The antibody- coated magnetic 4 particles are used to concentrate Salmonella Group D1bacteria present within an 5 enriched sample making confirmation of the presumptive positive result much more 6 robust and easier to interpret. Essentially, the coated magnetic particles are added to a 7 presumptive positive enrichment. If SE is present, it will bind to the magnetic particles 8 via the coated antibody. A magnet is then used to concentrate the bound, coated 9 magnetic particles and the remaining enrichment is discarded leaving only magnetic 10 particles bound to the Salmonella Group D1serovars present in the enrichment. 11 Confirmation procedures are then continued with the concentrated sample. 12 13

4. General Information 14 15

Salmonellosis is the most commonly occurring foodborne illness in humans and is 16 caused by the ingestion of Salmonella-contaminated food. The Centers for Disease 17 Control and Prevention (CDC) estimated that 1.4 million cases of Salmonellosis occur 18 annually in the U.S. and results in over 500 deaths (1). Salmonella Enteritidis (SE) is 19 one of the most frequently reported Salmonella serotypes associated with human illness. 20 In 2009, SE was the leading reported cause of Salmonella infections; accounting for 21 19.2% of the serotypes isolated (2). Most SE exposures have historically been 22 associated with the consumption of table (shell) eggs however recently, SE infection in 23 broiler chickens has been on the rise (3). It is estimated that approximately 47 billion 24 shell eggs are consumed annually in the US and that 2.3 million of those are 25 contaminated with SE (4). Due to this incidence of SE found in shell eggs and the large 26 number of shell eggs consumed in the US, the U.S. Food and Drug Administration 27 (FDA) has implemented regulations entitled “Prevention of Salmonella Enteritidis in 28 Shell Eggs During Production, Storage and Transportation; Final Rule” 21 CFR Parts 16 29 and 118 (5). The effective date of the Final Rule was July 9, 2009 with implementation 30 beginning July 9, 2010 for most egg farms. Part of the Final Rule requires egg farms to 31 implement an egg layer house environmental drag swab testing program. If SE is found, 32 egg pools (representing a total of 1000 eggs from a single days production at that 33 facility) must be tested or, alternatively, the eggs diverted for the life of the flock to 34 processing (e.g. pasteurization). Under the egg testing option, if a positive egg test 35 result occurs, the eggs must be diverted to processing for the life of the flock or until 4 36 egg pools in a row, at 2 week intervals, are found negative. At that time, the eggs from 37 that flock may be sold as shell eggs. Under the Final Rule, both environmental drag 38 swab testing and egg pool testing are required by Federal regulation. The test methods 39 under consideration here offer advantages over the existing cultural reference methods in 40 ease-of-use and time-to-result. This latter feature is particularly important for egg 41 testing when eggs are being diverted to processing pending the test result. 42

43 5. Test Kit Information 44 45


5.1 Kit Name.— RapidChek SELECTTM

Salmonella Enteritidis Test System; 1 RapidChek CONFIRM

TM Salmonella Enteritidis Immunomagnetic Separation (IMS) 2

Kit 3 5.2 Catalog Number.— 7000220; 7000225 4 5.3 Ordering Information.— 5

5.3.1 USA.— SDIX, 111 Pencader Drive, Newark, DE 19702. Phone: 800-544-6 8881. Fax: 302-456-6782. E-mail: [email protected]. 7

8 5.4 Test Kit Reagents and Preparation 9

10 5.4.1 7000220: 11

5.4.1.1 RapidChek SELECTTM

Salmonella Enteritidis Test Strips 12

5.4.1.2 Transfer pipettes (100 L) 13 5.4.1.3 Test Tubes 14 5.4.1.4 User Guide 15 5.4.1.5 RapidChek SELECT

TM Salmonella Primary Media. 16

Twenty (20) grams of primary media base was dissolved in 1 liter of 17 deionized water and autoclaved (121°C, 15 min) or filter sterilized. 18 Alternatively, media was rehydrated in sterile deionized water. In 19 this manner, rehydrated media was used within 3 hours of 20 preparation. For chicken carcass rinsates, 40 grams of primary 21 media base was dissolved in 1 liter of deionized water and autoclaved 22 or filter sterilized. Alternatively, media base was rehydrated in sterile 23 deionized water. In this manner, rehydrated media was used within 3 24 hours of preparation. 25


Salmonella Supplement. Just prior to use, 10 26 mL of supplement was added per liter of primary media base. The 27 media was shaken to mix. For chicken carcass rinsates, 20 mL of 28 supplement was added per liter of primary media base and shaken to 29 mix. 30


Salmonella Secondary Media. Secondary 31 media (73.5 g) was reconstituted in 1 liter of deionized water and 32 boiled with stirring. Alternatively, media was rehydrated in sterile 33 deionized water. In this manner, rehydrated media was used within 3 34 hours of preparation. 35

36 5.4.2 7000225: 37

5.4.2.1 Salmonella Group D1-Specific Immunomagnetic Separation (IMS) 38 Beads. The IMS beads are added to the enriched sample at a rate of 39 0.05 mL IMS beads per milliliter of sample. 40

5.4.2.2 Phosphate-buffered Saline-Tween (PBS-T) Packet (Sigma Cat No. 41 P3563). The contents of the packet was dissolved in 1 liter of 42 deionized water. 43

5.4.2.3 Sample Tubes (2 mL) 44 5.4.2.4 User Guide 45

46


6. Additional Supplies and Reagents Preparation 1 2

6.1 Buffered Peptone Water (BPW) - (Difco Becton Dickinson). Twenty (20) 3 grams of media was dissolved in one liter of deionized water and autoclaved for 4 15 min at 121

oC. 5

6 6.2 Trypticase soy broth (TSB) - (Difco Becton Dickinson). Thirty (30) grams of 7

media was dissolved in one liter of deionized water and autoclaved for 15 min at 8 121

oC. The TSB was supplemented with 35 mg/L ferrous sulfate for use with 9

the FDA-BAM reference method for shell eggs. 10 11

6.3 Tetrathionate (TT) Broth – (Difco Becton Dickinson). 46 g of media was added 12 to one liter of purified water and brought to a boil. It was allowed to cool to 13

below 60 C prior to dispensing into sterile, 10 mL test tubes. Just prior to use, 14 0.2 mL of iodine-iodide solution (6 g iodine and 5 g potassium iodide in 40 mL 15 deionized water) and 0.1 mL of brilliant green solution (0.1% (w/v)) was added 16 to each tube containing 10 mL of media base. 17

18 6.4 Tetrathionate (Hajna) Broth – (Difco Becton Dickinson). 91.5g of media was 19

added to one liter of deionized water and brought to a boil. It was allowed to 20

cool to below 50 C prior to dispensing into sterile, 10 mL test tubes. Just prior 21 to use, 0.4 mL of iodine-iodide solution (5 g iodine and 8 g potassium iodide in 22 40 mL deionized water) was added to each tube containing 10 mL of media 23 base. 24

25 6.5 Rappaport Vassilidis broth (RV) (for FDA-BAM procedures (poultry drag 26

swabs and egg pools))– (Difco Becton Dickinson). For these applications, RV 27 broth was made from individual ingredients. Five (5) g tryptone, 8 g sodium 28 chloride, and 1.6 g potassium phosphate monobasic were dissolved in 1 L of 29 distilled water were combined. Four hundred grams of magnesium chloride 30 hexahydrate was dissolved in 1 L of distilled water. Malachite green oxalate 31 (0.4 g) was dissolved in 100 mL of distilled water. Complete media was made 32 by combining 1000 mL broth base, 100 mL magnesium chloride solution, and 33 10 mL malachite green oxalate solution (total volume of complete medium is 34 1110 mL). The liquid media was dispensed (10 mL) into culture tubes and 35 autoclaved for 15 min at 115

oC. 36

37 6.6 Rappaport Vassilidis R10 broth (RV) (for FSIS procedure (carcass rinsates))– 38

(Difco Becton Dickinson). To 1 L of deionized water, 26.6 g of media was 39 added and heated gently to dissolve. The liquid media was dispensed into 10 40

mL tubes and autoclaved at 116 C for 15 min. 41 42

6.7 Brilliant green sulfa agar (BGS) – (Remel) Fifty-eight (59) grams of media was 43 added to 1 liter of deionized water, boiled for 1 min, and autoclaved for 15 44 minutes at 121

oC. 45

46


6.8 Brilliant green novobiocin agar (BGN). (Difco Becton Dickinson) Fifty-eight 1 (58) grams of brilliant green agar was dissolved in 1 L of deionized water, 2 boiled for 1 min, and autoclaved for 15 minutes at 121

oC. To this was added 1 3

mL of a 20 mg/mL solution of novobiocin. 4 5

6.9 Xylose Lysine Tergitol 4 agar (XLT4) - (Difco Becton Dickinson) Fifty-nine 6 (59) grams of media was added to 1 liter of deionized water. Then, 4.6mL of 7 Tergitol 4 (Niaproof 4, Sigma) was added and the liquid brought to a boil for 1 8 min. 9

10 6.10 Xylose Lysine Deoxycholate agar (XLD) - (Difco Becton Dickinson) Fifty-five 11

(55) grams of media was added to 1 liter of deionized water and the liquid 12 brought to a boil for 1 min. 13

14 6.11 Bismuth sulfite agar (BS) - (Difco Becton Dickinson) Fifty-two (52) grams of 15

media was added to 1 liter of deionized water and the liquid brought to a boil. 16 17 6.12 Hektoen Enteric agar (HE) - (Difco Becton Dickinson) Seventy-six (76) grams 18

of media was added to 1 liter of deionized water and the liquid brought to a boil. 19 20 6.13 Triple Iron Sugar Agar Slant (TSI) - (Becton Dickinson Biosciences) 21

22 6.14 Lysine Iron Agar Slant (LIA) – (Becton Dickinson Biosciences) 23

24 6.15 API 20E kits for identification of Enterobacteriaceae – (BioMerieux) 25

26 6.16 Salmonella Group D1 antisera (Factors 1, 9, 12) (Difco Becton Dickinson) 27

28 7. Apparatus 29 30

7.1 Stomacher bags with filter 31 32

7.2 Stomacher bags without filter 33 34

7.3 Inoculating needles and loops 35 36

7.4 Vortex mixer 37 38

7.5 Stationary incubator – Capable of holding temperature at 42oC+/- 2.0 for 20-24 39

hours. 40 41

7.6 Stationary incubator – Capable of holding temperature at 35oC +/-2.0 for 20-24 42

hours. 43 44

7.7 Stomacher – Seward 400 Circulator (Seward, London, England) - for thorough 45 mixing of food samples in enrichment broth. 46


1 7.8 Top loading balance – To weigh 25 g ( + 0.1g) test samples. 2

3 7.9 Magnetic Separation Rack- (e.g. Promega Corp. Part number CD4002) 4

5 7.10 Rocking Mixer– Barnstead Thermolyne Speci-Mix M26125 (Barnstead 6

Thermolyne, Dubuque, IA), Fixed Speed: 18 rpm. 7 8

7.11 Circulating water bath capable of holding temperature at 42 +/- 0.2 oC. 9

10 8. Standard Reference Materials 11 12

Salmonella Enteritidis ATCC 13076, Salmonella Enteritidis ARS 11, Salmonella 13 Enteritidis ARS 12, and Salmonella Kentucky ATCC 9263 were used in various aspects 14 of the study. Salmonella Enteritidis ATCC 13076 and Salmonella Kentucky ATCC 15 9263 were obtained from the American Tissue Culture Collection (ATCC, Gaithersburg, 16 MD). Salmonella Enteritidis strains ARS 11 and ARS 12 were obtained from the 17 Eastern Regional Research Center, USDA-ARS, Wyndmoor, PA. 18

19 Additional bacterial strains were used in the Inclusivity and Exclusivity studies and are 20 indicated in Tables 1 and 2. 21

22 9. Safety Precautions 23 24

Salmonella is a human pathogen. All test strips, pipettes and media were 25 decontaminated by autoclave, bleach, etc., in accordance with local, state and federal 26 regulations. 27 28

10. General Preparation 29 30

10.1 All incubators and circulating water baths were calibrated and monitored using 31 NIST-certified thermometers. 32

33 11. Sample Preparation 34 35

11.1 Poultry house drag swabs were obtained from an egg facility in Maine. The hen 36 house was occupied by approximately 20,000 pullets (16 weeks old) at the time of 37 sampling. The drag swab samples were collected from the manure pits below the 38 cage banks according to methods described in Appendix 3.3 “Environmental 39 Sampling and Detection of Salmonella In Poultry Houses” US FDA, October 2008. 40 One lot of one hundred and twenty-five (125) drag swab samples were collected and 41 shipped to SDIX for next day delivery. Upon receipt, 60 drags were subsequently 42 shipped to the independent laboratory for next day delivery. The remaining swabs 43 were used for the internal studies. Drag swabs were used within 48 h of sampling 44 and maintained at approximately 4 °C throughout shipping and storage. 45

11.2 Shell eggs were obtained from a local wholesale food provider. 46


11.3 Chicken broiler carcasses were obtained from a local grocery store. 1 2

12. Interpretation and Test Result Report 3 4

The Mantel-Haenszel chi-square test for unmatched test portions was used to determine 5 whether two methods were equivalent. A Chi-Square value of less than 3.84 indicates 6 no significant difference (p<0.05) in the numbers of positive test portions given by the 7 two methods being compared. The formula for determining the Mantel-Haenszel chi-8 square value is: 9 10 11

(N-1)(AF – (B+C+D)E)2 12

X2 = ----------------------------------------------- 13

(A+B+C+D)(A+E)(B+C+D+F)(E+F) 14 15

Where N = A + B + C + D + E + F 16 17

Table 2. Data Table for Chi Square Analysis of Independent Samples

Confirmed+ Confirmed -

Alternative

Method

Presumptive + A B

Presumptive - C D

Reference Method E F

18 19 Only presumptive positive results that confirm positive are considered as positive for the 20 alternative method. All other results (presumptive positives that confirm negative, 21 presumptive negatives that confirm negative and presumptive negatives that confirm 22 positive) are considered as negative for the alternative method. 23 24

25 13. Internal Validation Studies 26

27 13.1 Poultry House Drag Swab Method Comparison Study 28

29 Poultry house drag swabs were collected using standard procedures outlined in 30 “Environmental Sampling and Detection of Salmonella In Poultry Houses” (US 31 FDA, October 2008) from an egg layer facility with no history of SE 32 contamination. For spiking, a single colony isolate of Salmonella Enteritidis 33 ARS 11 (originally isolated from poultry feces) was grown in trypticase soy 34 broth incubated at 37 ºC for 24 h. Viable cell enumeration of the inoculum was 35 accomplished by dilution plate counting on trypticase soy agar plates 36 supplemented with 0.6% yeast extract. Individual drag swabs were inoculated at 37 a target level of 3 CFU/swab. Two (2) sets of 5 unspiked and 20 spiked drag 38

swab samples were acclimated at 4 C for 48 h, tested by either the SDIX 39 method or the FDA BAM reference method, and the results compared. 40


1 13.1.1 Methodology 2 3

13.1.1.1 FDA Method – Environmental Sampling and Detection of Salmonella 4 in Poultry Houses (6) 5

6 One hundred (100) mL of buffered peptone water (BPW) was added 7 to the sterile whirl-pak bag containing the drag swab sample. The 8 sample was shaken and incubated 35 ± 2ºC for 24 ± 2 h. Then, 1 mL 9 of the sample was transferred into 10 mL TT broth and incubated at 10 43.0 ± 0.2ºC for 24 ± 2 h in a circulating water bath. In addition, 0.1 11 mL was transferred into 10 mL RV broth and incubated at 42.0 ± 12 0.2ºC for 24 ± 2 h in a water bath. Following the secondary 13 enrichment, a 10 µL loop of each secondary enrichment broth was 14 struck to XLT4 and BGN selective agar plates. The plates were 15 incubated at 35°C and examined 24 ±2 h. Up to 5 typical colonies 16 from each set of plates were transferred to TSI and LIA slants and 17 incubated 35 ± 1ºC for 24 ± 2 h. A minimum of 1 positive TSI slant 18 was subjected to slide agglutination serology testing for Salmonella 19 Group D1. If positive, then the isolate was identified biochemically 20 using BioMerieux API20E (Official Method 978.24). 21

22 13.1.1.2 RapidChek SELECT

TM Salmonella Enteritidis Test System 23

24 One hundred (100) mL of pre-warmed (42ºC) supplemented 25 RapidChek SELECT

TM primary media was added to each sample bag 26

containing the swab, hand massaged for 30 sec, and incubated at 27 42±2°C for 16-22 hr. Then, 0.2 mL of the primary enrichment broth 28 was transferred to a tube containing 2 mL pre-warmed (42ºC) 29 RapidChek secondary media and incubated 16-22 h at 42ºC. Then, 30 the tubes were gently shaken, the lateral flow test strip was inserted 31 into the tube and developed for 10 min. The results were recorded 32 (Positive – two red lines, Negative – one red line). All samples were 33 confirmed according to the FDA-BAM method described above using 34 the RapidChek SELECT

TM secondary enrichment broth. 35

36 In addition, all samples positive by the RapidChek SELECT

TM 37

Salmonella Enteritidis test strip were tested with the RapidChek 38 CONFIRM Salmonella Enteritidis IMS method. For this, the working 39 stock of monoclonal antibody-magnetic particle reagent was re-40 suspended by repeated inversion of the vial. One (1) mL of enriched 41 sample was transferred to the a 2 mL polypropylene centrifuge tube. 42 Then, 0.05 mL of the IMS beads was added to the enriched sample in 43 the tube, vortexed briefly to mix and incubated at room temperature 44 with rocking for 15 minutes. The sample tube was placed on a 45 magnetic separation rack for 5 minutes after which time the liquid 46


was removed from the sample. The sample tubes were removed from 1 the magnetic rack and 1 mL of wash solution (PBS-T) was added to 2 the tube. It was vortexed briefly to mix and placed on the magnetic 3 separation rack for 5 minutes after which time the liquid was removed 4 from the sample. The wash step was repeated for a total of 5 washes. 5 After the final wash step, the sample was reconstituted with 0.1 mL of 6 wash solution and vortexed briefly to mix. Selective agar plates 7 (XLT4 and BGN) were struck with a 10 µL loop of the purified 8 sample. Plates were incubated at 35°C and examined after 24 ±2 h. 9 Typical Salmonella colonies were confirmed as described above. 10

11 13.1.2 Results 12 13

The results from the study are shown in Table 1. Thirteen (13) samples were 14 positive with the RapidChek SELECT

TM SE test strip. All of the 15

presumptive positives samples confirmed as Salmonella Group D1 positive. 16 None of the test strip-negative samples (12) were culturally-positive by the 17 FDA-BAM confirmation method. The FDA-BAM reference method 18 detected 4 positive samples. This gave an accuracy of 325% indicating that 19 3.25 times more positives were found with the RapidChek SELECT

TM SE 20

method than with the reference method. The chi-square value was 8.08, 21 indicating the RapidChek method is significantly more sensitive than the 22 reference method for the detection of SE in poultry house drag swabs. 23

24 13.2 Egg Pool Method Comparison Study 25 26

Shell eggs were purchased from a local wholesale food distributor. 27 Approximately 1200 eggs were disinfected using established FDA-BAM 28 procedures (70% ethanol/iodide/iodine solution). Nine hundred (900) eggs were 29 cracked and the contents pooled. For spiking, a single colony isolate of 30 Salmonella Enteritidis ATCC 13076 (a clinical isolate) was grown in trypticase 31 soy broth incubated at 37 ºC for 24 h. Viable cell enumeration of the inoculm was 32 accomplished by dilution plate counting on trypticase soy agar plates 33 supplemented with 0.6% yeast extract. The egg pool was spiked at a target level 34 of 2.4 CFU/L (approximate volume of 20 egg pool). For the negative control 35 samples, 300 disinfected eggs were cracked and pooled. One (1) liter pools were 36 aliquoted into sterile bags and acclimated for 48 h at 4°C prior to analysis by the 37 SDIX and FDA-BAM cultural reference methods. At the time of analysis, 38 following acclimation, an aerobic plate count was conducted on the negative pool 39 and most probable number (MPN) analysis was conducted on the spiked pool. 40


13.2.1.1 FDA-BAM Cultural Reference Method (7) 44 45


For the FDA-BAM method, following acclimation at 4°C for 48 h, 1 eggs pools were held at room temperature (20-24°C) for 96 ± 2 h. 2 After that time, a 25 mL portion from each sample was removed and 3 added to 225 mL of sterile trypticase soy broth (TSB) supplemented 4 with ferrous sulfate (35 mg/L) and mixed well by swirling. This 5 sample was incubated 60 ± 5 min at room temperature. The sample 6 was mixed by swirling and the pH was adjusted to 6.8 ± 0.2. The 7 sample was incubated 24 ± 2 h at 35°C. Then, 0.1 mL was 8 transferred to 10 mL Rappaport-Vassiliadis (RV) broth (prepared 9 from individual ingredients) and 1 mL to 10 mL TT broth. The RV 10

was incubated at 42 0.2°C for 24 2 h and the TT broth at 35 ± 11

0.2 C for 24 2 h. After this incubation, the samples were mixed and 12

then struck (10 l) onto BS, XLD and HE agars. The plates were 13

incubated at 35 C for 24 2 h. Up to 5 typical colonies were 14

transferred to TSI and LIA slants and incubated at 35 C for 24 2 h. 15 A minimum of 1 positive TSI slant was subjected to slide 16 agglutination serology testing for Salmonella Group D1. If positive, 17 then the isolate was identified biochemically using BioMerieux 18 API20E (Official Method 978.24). 19

20 13.2.1.2 RapidChek SELECT

TM SE Test System 21

22 Following acclimation at 4°C for 48 h, to each egg pool was added 23 200 mL pre-warmed (42ºC) supplemented RapidChek SELECT

TM 24

primary media. The sample was mixed well and incubated at room 25 temperature (23ºC) for 40 h. At that time, 0.1 mL of the primary 26 enrichment broth was added to a tube containing 1 mL pre-warmed 27 (42ºC) RapidChek SELECT

TM secondary media. The tubes were 28

incubated 6-8 h at 42ºC. The tubes were gently shaken and a 29 RapidChek SELECT

TM SE test strip was added. The test strip was 30

developed for 10 min and the result recorded. The results were 31 recorded (Positive – two red lines, Negative – one red line). All 32 samples were confirmed according to the FDA-BAM method 33 described above using the RapidChek SELECT

TM secondary 34

enrichment broth. 35 36

13.2.2 Results 37 38

The results from the study are shown in Table 2. Seventeen (17) samples 39 were positive with the RapidChek SELECT


presumptive positives samples confirmed as Salmonella Group D1 positive. 41 None of the test strip-negative samples (8) were culturally-positive. The FDA-42 BAM reference method detected 15 positive samples. This gave an accuracy of 43 113% indicating that the RapidChek Select SE method gave nearly the same 44 number of positives as the reference method. The chi-square value was 0.609, 45 indicating the RapidChek method is equivalent to the FDA-BAM method for the 46


detection of SE in egg pools. Furthermore, the RapidChek Select SE method 1 gave a negative result after 48 h (2 days) whereas the FDA-BAM method 2 required 168 h (7 days) for a negative result. 3

4


Table 1. Results from the Poultry House Drag Swab Method Comparison Study-Internal Validation. 1

2 aMantel-Haenszel Chi-square analysis.

bSensitivity Rate = (No. of test method presumptive positives)/(No. of test method confirmed positives) x 100. 3

cFalse Negative Rate = 100 - Sensitivity Rate.

dSpecificity Rate = (No. of test method negatives)/(No. of confirmed test method negatives) x 100. 4

eFalse Positive Rate = 100 - Specificity Rate.

fAccuracy = (No. of test method positives)/(No. of reference method positives) x 100. 5

6

7 Table 2. Results from the Egg Pool Method Comparison Study-Internal Validation. 8







13

14 Table 3. Results from the Chicken Carcass Rinsate Method Comparison Study-Internal Validation. 15







Matrix Analyte MethodNumber of

Samples

Inoculation

Level,

CFU/sample

Presumptive

Positives

Confirmed

Positives

Reference

Method

Positives

Chi squarea

Sensitivity

Rateb

False

Negative

Ratec

Specificity

Rated

False Positive

Ratee Accuracy

f

5 0 0 0 0

20 3 13 13 4

Poultry

House Drag

Swabs

S. Enteritidis

ARS 11

RapidChek

SELECT8.070 100.0% 0.0% 100.0% 0.0% 325.0%


Samples

MPN,

CFU/sample

Presumptive

Positives

Confirmed

Positives

Reference

Method

Positives

Chi squarea

Sensitivity

Rateb

False

Negative

Ratec

Specificity

Rated

False Positive

Ratee Accuracy

f

5 0 0 0 0

20 <3 17 17 15

Egg PoolsS. Enteritidis

ATCC 13076

RapidChek

SELECT0.609 100.0% 113.3%0.0% 100.0% 0.0%


Samples

Inoculation

Level,

CFU/sample

Presumptive

Positives

Confirmed

Positives

Reference

Method

Positives

Chi squarea

Sensitivity

Rateb

False

Negative

Ratec

Specificity

Rated

False Positive

Ratee Accuracy

f

5 0 0 0 0

20 1 11 11 13

0.0% 100.0% 0.0% 84.6%

Chicken

Carcass

Rinsates

S. Enteritidis

ARS 11

RapidChek

SELECT0.406 100.0%


13.3 Chicken Carcass Rinsate Method Comparison Study 1 2

Whole (broiler) chicken carcasses (25) were obtained from a local grocery store. 3 They were asceptically drained of excess liquid and transferred to a large sterile 4 bag. For spiking, a single colony isolate of Salmonella Enteritidis ARS 11 5 (originally isolated from poultry feces) was grown in trypticase soy broth 6 incubated at 37 ºC for 24 h. Viable cell enumeration of the inoculum was 7 accomplished by dilution plate counting on trypticase soy agar plates 8 supplemented with 0.6% yeast extract. Individual carcasses were inoculated at a 9 target level of 13.3 CFU/carcass or 1 CFU/30 mL rinsate (analytical unit). Four 10 hundred (400) mL of BPW was poured into the cavity of the carcass contained in 11 the bag. The bird was rinsed inside and out with a rocking motion for one minute. 12


13.3.1.1 USDA-FSIS Cultural Reference Method (8) 16 17

Thirty (30) mL of the sample rinse fluid obtained above was 18 transferred to a sterile stomacher bag followed by 30 mL of sterile 19 BPW and mixed well. The sample was incubated at 35 ± 2°C for 20-20 24 h. Then, 0.5 ± 0.05 mL of this was transferred into 10 mL TT 21 (Hajna) broth and 0.1 ± 0.02 mL into 10 mL mRV broth and 22 incubated at 42 ± 0.5°C for 22-24 h. Following incubation, the 23 broths were struck (10 µL) to XLT4 and BGS agar plates. The plates 24 were incubated at 35 ± 2°C for 18-24 h at which time typical colonies 25 were picked for confirmation. If negative, plates were re-incubated 26 for an additional 18-24 h and re-examined. Up to 5 typical colonies 27

were transferred to TSI and LIA slants and incubated at 35 C for 24 28 2 h. A minimum of 1 positive TSI slant was subjected to slide 29 agglutination serology testing for Salmonella Group D1. If positive, 30 then the isolate was identified biochemically using BioMerieux 31 API20E (Official Method 978.24). 32

33 13.3.1.2 RapidChek SELECT SE Test System 34 35

Thirty (30) mL of the sample rinse fluid obtained above was 36 transferred to a sterile stomacher bag followed by 30 mL of pre-37 warmed (42ºC) 2X RapidChek SELECT

TM primary media containing 38

2X supplement and mixed well. The sample was incubated at 39 42±2°C for 16-22 hr. Then, 0.1 mL of the primary enrichment broth 40 was transferred to a tube containing 1 mL pre-warmed (42ºC) 41 RapidChek SELECT

TM secondary media and incubated 16-22 h at 42

42ºC. Then, the tubes were gently shaken, the lateral flow test strip 43 was inserted into the tube and developed for 10 min. The results were 44 recorded (Positive – two red lines, Negative – one red line). All 45 samples were confirmed according to the USDA-FSIS method 46


described above using the RapidChek SELECTTM

secondary 1 enrichment broth. 2

3 13.3.2 Results 4 5

The results from the study are shown in Table 3. Eleven (11) samples were 6 positive with the RapidChek SELECT

TM SE test strip. All of the presumptive 7

positives samples confirmed as Salmonella Group D1 positive. None of the test 8 strip-negative samples (14) were culturally-positive. The USDA-FSIS reference 9 method detected 13 positive samples. This gave an accuracy of 85% indicating 10 that the RapidChek SELECT

TM SE method gave nearly the same number of 11

positives as the reference method. The chi-square value was 0.406, indicating 12 the RapidChek method is equivalent to the USDA-FSIS cultural reference 13 method for the detection of SE in chicken carcass rinsates. 14

15 13.4 Test Strip Inclusivity Study 16 17

13.4.1 Methodology 18 Salmonella single colony isolates were cultured in 10 mL of RapidChek 19

SELECTTM

primary enrichment media for 16-22 hr at 42 C. Then, 0.1 mL 20 of the primary broth was transferred to 1 mL of RapidChek SELECT

TM 21

secondary enrichment media. This was incubated at 42 C for 16-22 hr. 22 Following enrichment, a test strip was inserted into the tube and developed 23 for 10 min after which time the test strip was read and interpreted as 24 previously described. 25

26 13.4.2 Results 27 28

The results from the test inclusivity study are shown in Table 4. Eighty-two 29 (82) Salmonella Group D1 strains including 63 SE strains were grown in the 30 complete RapidChek SELECT

TM Salmonella media system and tested with 31

the test strip. All of the Salmonella Group D1 strains gave a positive result 32 with the method. This indicated a method sensitivity of 100%. 33


Table 4. Results from the Test Strip Inclusivity Study. 1

2

Sample Number Serovar Strain Number

RapidChek Select SE

Test Strip Result Sample Number Serovar Strain Number

RapidChek Select SE

Test Strip Result

1 Salmonella Enteritidis ARS 11 + 43 Salmonella Enteritidis ISU-18-4h +

2 Salmonella Enteritidis ARS 12 + 44 Salmonella Enteritidis ISU-18-5d +

3 Salmonella Enteritidis M1 BGA 164-93 + 45 Salmonella Enteritidis ISU-18-6n +

4 Salmonella Enteritidis Tyson 22 + 46 Salmonella Enteritidis ISU-18-9f +

5 Salmonella Enteritidis ATCC 13076 + 47 Salmonella Enteritidis ISU-18-10g +


7 Salmonella Enteritidis var. Jena ATCC 49221 + 49 Salmonella Enteritidis ISU-20-19i +

8 Salmonella Enteritidis var. Jena ATCC 49222 + 50 Salmonella Enteritidis ISU-20-32m +

9 Salmonella Enteritidis var. Jena ATCC 49223 + 51 Salmonella Enteritidis ISU-20-33n +

10 Salmonella Enteritidis var. Essen ATCC 49218 + 52 Salmonella Enteritidis ISU-20-36p +

11 Salmonella Enteritidis var. Essen ATCC 49219 + 53 Salmonella Enteritidis ISU-20-35q +

12 Salmonella Enteritidis var. Essen ATCC 49220 + 54 Salmonella Enteritidis ISU-20-36r +

13 Salmonella Enteritidis var. Danysz ATCC 49217 + 55 Salmonella Enteritidis ISU-21-5f +

14 Salmonella Enteritidis var. Chaco ATCC 49214 + 56 Salmonella Enteritidis ISU-22-5a +

15 Salmonella Enteritidis var. Chaco ATCC 49215 + 57 Salmonella Enteritidis ISU-22-6b +

16 Salmonella Enteritidis ISU-1-2P + 58 Salmonella Enteritidis ISU-23-5e +

17 Salmonella Enteritidis ISU-1-4K + 59 Salmonella Enteritidis ISU-23-5h +

18 Salmonella Enteritidis ISU-1-6J + 60 Salmonella Enteritidis ISU-24-3a +

19 Salmonella Enteritidis ISU-1-38s + 61 Salmonella Enteritidis ISU-24-4b +

20 Salmonella Enteritidis ISU-1-78t + 62 Salmonella Enteritidis ISU-24-5c +

21 Salmonella Enteritidis ISU-5-4j + 63 Salmonella Enteritidis ISU-25-1f +

22 Salmonella Enteritidis ISU-6-19i + 64 Salmonella Dublin ISU-2-1a +


24 Salmonella Enteritidis ISU-7-6f + 66 Salmonella Dublin ISU-4-1a +

25 Salmonella Enteritidis ISU-8-27e + 67 Salmonella Berta ISU-16-2b +

26 Salmonella Enteritidis ISU-8-13a + 68 Salmonella Berta ISU-16-3i +

27 Salmonella Enteritidis ISU-9-13e + 69 Salmonella Berta ISU-16-7j +

28 Salmonella Enteritidis ISU-10-3e + 70 Salmonella Berta ISU-16-10f +

29 Salmonella Enteritidis ISU-10-9g + 71 Salmonella Berta ISU-16-12k +

30 Salmonella Enteritidis ISU-10-13d + 72 Salmonella Javiana ATCC 10721 +

31 Salmonella Enteritidis ISU-10-13p + 72 Salmonella Panama Tyson 3 +

32 Salmonella Enteritidis ISU-11-2a + 73 Salmonella Pullorum ATCC 9120 +

33 Salmonella Enteritidis ISU-11-2f + 74 Salmonella Pullorum ATCC 19945 +

34 Salmonella Enteritidis ISU-12-39s + 75 Salmonella 9,12:nonmotile ISU-10-3a +

35 Salmonella Enteritidis ISU-12-42e + 76 Salmonella 9,12:nonmotile ISU-10-5b +

36 Salmonella Enteritidis ISU-12-53p + 77 Salmonella 9,12:nonmotile ISU-10-9c +

37 Salmonella Enteritidis ISU-13-10f + 78 Salmonella 9,12:nonmotile ISU-10-19i +

38 Salmonella Enteritidis ISU-13-11e + 79 Salmonella 9,12: poorly motile ISU-10-5"o" +

39 Salmonella Enteritidis ISU-14-8g + 80 Salmonella 9,12: poorly motile ISU-10-9n +

40 Salmonella Enteritidis ISU-15-2h + 81 Salmonella 9,12: poorly motile ISU-10-13h +

41 Salmonella Enteritidis ISU-17-43h + 82 Salmonella 9,12: poorly motile ISU-10-15m +

42 Salmonella Enteritidis ISU-18-3b +


13.5 Test Strip Exclusivity Study 1 2

13.5.1 Methodology 3 Single colony isolates of non-Salmonellae as well as non-Group D1 4 Salmonella bacteria were cultured in 10 mL of Tryptic soy broth (TSB) that 5

was incubated at 37 C for 24 h. One (1) mL of the broth culture was 6 transferred to a 12 x 75 mm tube and a test strip inserted into the tube. The 7 test strip was read and interpreted as previously described. 8

9 13.5.2 Results 10 11

The results from the test exclusivity study are shown in Table 5. 12 13 Table 5. Results from the Test Strip Exclusivity Study. 14

15 16

Bacteria Strain Number

RapidChek Select SE

Test Strip Result

Salmonella Typhimurium (B) ATCC 14028 -

Salmonella Heidelberg (B) WVU 5F114 -

Salmonella Montevideo (C1) ARS 32 -

Salmonella Thompson (C1) ARS 15 -

Salmonella Hadar (C2) ATCC 51956 -

Salmonella Kentucky (C3) ATCC 9263 -

Salmonella Albany (C3) ATCC 51960 -

Salmonella Maarsen (D2) ATCC 15793 -

Salmonella Muenster (E1) WVU 5F22 -

Salmonella Illinois (E3) ATCC 11646 -

Salmonella Senftenberg (E4) WVU 6F11 -

Salmonella Abaetetuba (F) ATCC 35640 -

Salmonella Poona (G1) DSM 109 -

Salmonella Cubana (G2) ATCC 12007 -

Salmonella Pomona (M) ATCC 10729 -

Bacillus subtilis ATCC 6633 -

Aeromonas veronii ATCC 51106 -

Citrobacter koseri ATCC 27026 -

Citrobacter freundii ATCC 8090 -

Enterobacter cloacae ATCC 27508 -

Enterobacter aerogenes ATCC 15038 -

Escherichia coli ATCC 35218 -


Escherichia hermannii ATCC 55236 -


Klebsiella pneumoniae ATCC 29018 -


Proteus vulgaris ATCC 8427 -

Proteus mirabilis ATCC 4630 -

Serratia liquefaciens ATCC 27592 -

Vibrio parahaemolyticus ATCC 17802 -



Thirty-two ( 32) non-Salmonella Group D1 bacteria from 10 genera were 1 tested in the exclusivity study. This included 15 Salmonella from serogroups 2 other than D1. None of the test strains gave a positive response in the test 3 indicating a specificity of 100%. 4

5 13.6 Test Strip Ruggedness Studies 6 7

13.6.1 Test Strip Read Time 8 9 13.6.1.1 Methodology 10

11 Salmonella Enteritidis ATCC 13076 and Salmonella Enteritidis 12 ARS 11 single colony isolates were cultured in 10 mL of 13 RapidChek SELECT

TM primary enrichment media for 16-22 hr at 14

42 C. Then, 0.1 mL of the primary broth was transferred to 1 mL 15 of RapidChek SELECT

TM secondary enrichment media. This was 16

incubated at 42 C for 16-22 hr. The samples were then diluted 17 into RapidChek SELECT

TM secondary media to approximately 1 18

x 106 CFU/mL (approximately 1 log10 above the detection limit of 19

the method). Salmonella Kentucky ATCC 9263 was grown in 20 non-selective media (TSB) (approximately 1 x 10

9 CFU/mL). 21

Test strips were placed into replicate samples (5), removed from 22 the sample at various time points (5, 10, 20 min) and the test strip 23 was read and interpreted. 24

25 13.6.1.2 Results 26

27 The results are shown in Table 4. 28

29 Table 4. Results from the Test Strip Read Time Ruggedness Study. 30

31 32

There were no differences in the number of test strip positives 33 found when test strips were interpreted after 5, 10 (designated 34 read time), or 20 min in the sample. In addition, there were no 35 false positives found with the non-Salmonella Group D1 strain. 36 This suggested that the impact of test strip read time on the test 37 strip result is minimal. 38

39 13.6.2 Test Strip Sample Temperature 40

41

Test Level, CFU/mL 5 10 20

S. Enteritidis ATCC 13076 1 x 106

5/5 5/5 5/5

S. Enteritidis ARS 11 1 x 106

5/5 5/5 5/5

S. Kentucky ATCC 9263 ~1 x 109

(Neat) 0/5 0/5 0/5

Test Strip Result, # of Positives

Read Time, min


13.6.2.1 Methodology 1 2

Salmonella Enteritidis ATCC 13076 and Salmonella Enteritidis 3 ARS 11 single colony isolates were cultured in 10 mL of 4 RapidChek SELECT




incubated at 42 C for 16-22 hr. The samples were then diluted 8 into RapidChek SELECT

TM secondary media to approximately 1 9

x 106 CFU/mL (approximately 1 log10 above the detection limit of 10

the method). Salmonella Kentucky ATCC 9263 was grown in 11 non-selective media (TSB) (approximately 1 x 10

9 CFU/mL). 12

Replicate (5) 1 mL aliquots were left at room temperature or 13 placed in an incubator for 1 hr in order to further acclimate the 14 sample at various test temperatures (room temperature (20-25°C), 15 42°C, and 44°C). Test strips were placed into the samples while 16 maintained under the various conditions (i.e., in the respective 17 incubator). They were developed for 10 min, removed and 18 interpreted. 19

20 13.6.2.2 Results 21


24 Table 5. Results from the Test Strip-Sample Temperature Ruggedness 25 Study. 26

27 aRT, room temperature, 23-25°C 28 29

There were no differences in the number of test strip positives 30 found when test strips were tested on samples that varied in 31 temperature from room temperature (20-25°C) to 44°C. In 32 addition, there were no false positives found with the non-33 Salmonella Group D1 strain. This suggested that the impact of 34 sample temperature on the test strip result is minimal. 35

36 13.6.3 Test Strip Sample Volume 37

38 13.6.3.1 Methodology 39

40

Test Level, CFU/mL RTa

42 44


5/5 5/5 5/5


5/5 5/5 5/5


(Neat) 0/5 0/5 0/5


Test Temperature, °C


Salmonella Enteritidis ATCC 13076 and Salmonella Enteritidis 1 ARS 11 single colony isolates were cultured in 10 mL of 2 RapidChek SELECT




incubated at 42 C for 16-22 hr. The samples were then diluted 6 into RapidChek Select secondary media to approximately 1 x 10

6 7

CFU/mL (approximately 1 log10 above the detection limit of the 8 method). Salmonella Kentucky ATCC 9263 was grown in non-9 selective media (TSB) (approximately 1 x 10

9 CFU/mL). 10

Replicate (5) aliquots of each sample type varying in sample 11 volumes (0.75, 1.0, 2.0, and 2.25 mL) were tested. Test strips 12 were placed into the samples, developed for 10 min, removed and 13 interpreted. 14

15 13.6.3.2 Results 16


19 Table 6. Results from the Test Strip-Sample Volume Ruggedness Study. 20

21 22

There were no differences in the number of test strip positives 23 found when test strips were tested on samples varying in sample 24 volume. In addition, there were no false positives found with the 25 non-Salmonella Group D1 strain. This suggested that the impact 26 of sample volume on the test strip result is minimal. 27

28 13.7 Test Strip Stability Study 29 30

13.7.1 Methodology 31 32

Three lots of test strips in their final packaging (50 test strips/desiccant 33 canister) were placed into storage at various temperatures (4°C, room 34 temperature (20-25°C, recommended storage condition), 37°C (accelerated 35 stability), and 45°C (accelerated stability)). At 7 day intervals, 15 test strips 36 from each lot were removed from each storage condition and tested in 37 replicate (5) with replicate samples (5) of Salmonella Enteritidis ATCC 38 13076 and Salmonella Enteritidis ARS 11 at 1 x 10

6 CFU/mL (approximately 39

1 log10 above the detection limit of the test strip) in RapidChek Select 40

Test Level, CFU/mL 0.75 1.0 2.0 2.25


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


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


(Neat) 0/5 0/5 0/5 0/5


Test Volume, mL


secondary media and Salmonella Kentucky ATCC 9263 grown in non-1 selective media (TSB) and tested neat (approximately 1 x 10

9 CFU/mL). 2

3 13.7.2 Results 4 5

The results from this study are shown in Table 7. 6 7 Table 7. Results of the Accelerated Test Strip Stability Study 8

9 10

There were no differences in test strip results for up to 8 weeks (compared to 11 week 0) for any of the storage conditions. This demonstrated that the test 12 strip was very stable under accelerated stability conditions (up to 45°C). 13

14 13.8 Immunomagnetic Separation (IMS) Reagent Inclusivity Study 15 16


Single colony isolates of various Group D1 Salmonella were cultured in 10 19 mL of RapidChek SELECT


42 C. Then, 0.1 mL of the primary broth was transferred to 1 mL of 21 RapidChek SELECT

TM secondary enrichment media. This was incubated at 22

42 C for 16-22 hr. Following enrichment, 1 mL of the enriched sample was 23 subjected to IMS as described in Section 13.1.1.2. The samples (10 µL) were 24 struck to BGN and XLT4 plates. The plates were incubated at 35 ± 2°C for 25 18-24 h at which time 1 typical colony was picked for confirmation. Typical 26

colonies were transferred to TSI and LSI slants and incubated at 35 C for 24 27

2 h. The positive TSI slant was subjected to slide agglutination serology 28 testing for Salmonella Group D1. 29

30

Time,

weeksTest Strain

Test Level,

CFU/mL1 2 3 1 2 3 1 2 3 1 2 3

0 - - - 5/5 5/5 5/5 - - - - - -

1 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5

2 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5

3 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5

4 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5

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

6 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5

7 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5

8 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5

0 - - - 5/5 5/5 5/5 - - - - - -

1 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5

2 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5

3 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5

4 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5

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

6 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5

7 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5

8 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5

0 - - - 0/0 0/0 0/0 - - - - - -

1 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0

2 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0

3 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0

4 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0

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

6 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0

7 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0

8 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0

Salmonella

Kentucky

ATCC 9263

1 x 109

(Neat)

Salmonella

Enteritidis

ARS11

1 x 106

Salmonella

Enteritidis

ATCC 13076

1 x 106

Lot

4

Temperature, °C

Room Temperature 37 45


13.8.2 Results 1 2

The results of this study are shown in Table 8. Eighty-two (82) Salmonella 3 Group D1 strains including 63 SE strains were grown in the RapidChek 4 Select Salmonella media and tested with the IMS method followed by 5 independent serological colony confirmation. All of the Salmonella Group 6 D1 strains gave a positive result with the IMS confirmation procedure. 7

8 13.9 Immunomagnetic Separation (IMS) Reagent Exclusivity Study 9 10


Single colony isolates of non-Salmonellae as well as non-Group D1 13 Salmonella bacteria were cultured in 10 mL of Tryptic soy broth (TSB) 14

incubated at 37 C for 24 h. Following enrichment, 1 mL of the enriched 15 sample was subjected to IMS as described in Section 13.1.1.2. The samples 16 (10 µL) were struck to BGN and XLT4 plates. The plates were incubated at 17 35 ± 2°C for 18-24 h at which time 1 typical colony was picked for 18 confirmation. Typical colonies were transferred to TSI and LSI slants and 19

incubated at 35 C for 24 2 h. The positive TSI slant was subjected to slide 20 agglutination serology testing for Salmonella Group D1. 21

22 13.9.2 Results 23 24

The results of this study are shown in Table 9. 25


Table 8. Results from the Immunomagnetic Separation (IMS) Method Inclusivity Study. 1

2

Sample Number Serovar Strain Number

RapidChek Confirm SE

IMS Result Sample Number Serovar Strain Number

RapidChek Confirm SE

IMS Result

1 Salmonella Enteritidis ARS 11 + 43 Salmonella Enteritidis ISU-18-4h +

2 Salmonella Enteritidis ARS 12 + 44 Salmonella Enteritidis ISU-18-5d +

3 Salmonella Enteritidis M1 BGA 164-93 + 45 Salmonella Enteritidis ISU-18-6n +

4 Salmonella Enteritidis Tyson 22 + 46 Salmonella Enteritidis ISU-18-9f +



7 Salmonella Enteritidis var. Jena ATCC 49221 + 49 Salmonella Enteritidis ISU-20-19i +

8 Salmonella Enteritidis var. Jena ATCC 49222 + 50 Salmonella Enteritidis ISU-20-32m +

9 Salmonella Enteritidis var. Jena ATCC 49223 + 51 Salmonella Enteritidis ISU-20-33n +

10 Salmonella Enteritidis var. Essen ATCC 49218 + 52 Salmonella Enteritidis ISU-20-36p +

11 Salmonella Enteritidis var. Essen ATCC 49219 + 53 Salmonella Enteritidis ISU-20-35q +

12 Salmonella Enteritidis var. Essen ATCC 49220 + 54 Salmonella Enteritidis ISU-20-36r +

13 Salmonella Enteritidis var. Danysz ATCC 49217 + 55 Salmonella Enteritidis ISU-21-5f +

14 Salmonella Enteritidis var. Chaco ATCC 49214 + 56 Salmonella Enteritidis ISU-22-5a +

15 Salmonella Enteritidis var. Chaco ATCC 49215 + 57 Salmonella Enteritidis ISU-22-6b +

16 Salmonella Enteritidis ISU-1-2P + 58 Salmonella Enteritidis ISU-23-5e +

17 Salmonella Enteritidis ISU-1-4K + 59 Salmonella Enteritidis ISU-23-5h +

18 Salmonella Enteritidis ISU-1-6J + 60 Salmonella Enteritidis ISU-24-3a +

19 Salmonella Enteritidis ISU-1-38s + 61 Salmonella Enteritidis ISU-24-4b +

20 Salmonella Enteritidis ISU-1-78t + 62 Salmonella Enteritidis ISU-24-5c +

21 Salmonella Enteritidis ISU-5-4j + 63 Salmonella Enteritidis ISU-25-1f +



24 Salmonella Enteritidis ISU-7-6f + 66 Salmonella Dublin ISU-4-1a +

25 Salmonella Enteritidis ISU-8-27e + 67 Salmonella Berta ISU-16-2b +

26 Salmonella Enteritidis ISU-8-13a + 68 Salmonella Berta ISU-16-3i +

27 Salmonella Enteritidis ISU-9-13e + 69 Salmonella Berta ISU-16-7j +

28 Salmonella Enteritidis ISU-10-3e + 70 Salmonella Berta ISU-16-10f +

29 Salmonella Enteritidis ISU-10-9g + 71 Salmonella Berta ISU-16-12k +

30 Salmonella Enteritidis ISU-10-13d + 72 Salmonella Javiana ATCC 10721 +

31 Salmonella Enteritidis ISU-10-13p + 72 Salmonella Panama Tyson 3 +

32 Salmonella Enteritidis ISU-11-2a + 73 Salmonella Pullorum ATCC 9120 +

33 Salmonella Enteritidis ISU-11-2f + 74 Salmonella Pullorum ATCC 19945 +

34 Salmonella Enteritidis ISU-12-39s + 75 Salmonella 9,12:nonmotile ISU-10-3a +

35 Salmonella Enteritidis ISU-12-42e + 76 Salmonella 9,12:nonmotile ISU-10-5b +

36 Salmonella Enteritidis ISU-12-53p + 77 Salmonella 9,12:nonmotile ISU-10-9c +

37 Salmonella Enteritidis ISU-13-10f + 78 Salmonella 9,12:nonmotile ISU-10-19i +

38 Salmonella Enteritidis ISU-13-11e + 79 Salmonella 9,12: poorly motile ISU-10-5"o" +

39 Salmonella Enteritidis ISU-14-8g + 80 Salmonella 9,12: poorly motile ISU-10-9n +

40 Salmonella Enteritidis ISU-15-2h + 81 Salmonella 9,12: poorly motile ISU-10-13h +

41 Salmonella Enteritidis ISU-17-43h + 82 Salmonella 9,12: poorly motile ISU-10-15m +

42 Salmonella Enteritidis ISU-18-3b +


Table 9. Results from the Immunomagnetic Separation (IMS) Method 1 Exclusivity Study. 2

3 4

Thirty-two ( 32) non-Salmonella Group D1 bacteria from 10 genera were 5 tested in the IMS exclusivity study. This included 15 non-Group D1 6 Salmonella. None of the test strains gave a positive response with the IMS 7 confirmation procedure. 8

9 10

13.10 Immunomagnetic Separation (IMS) Reagent Ruggedness Study 11 12

13.10.1 Incubation Time 13 14

Bacteria Strain Number

RapidChek Confirm

SE IMS Result

Salmonella Typhimurium (B) ATCC 14028 -

Salmonella Heidelberg (B) WVU 5F114 -

Salmonella Montevideo (C1) ARS 32 -

Salmonella Thompson (C1) ARS 15 -

Salmonella Hadar (C2) ATCC 51956 -

Salmonella Kentucky (C3) ATCC 9263 -

Salmonella Albany (C3) ATCC 51960 -

Salmonella Maarsen (D2) ATCC 15793 -

Salmonella Muenster (E1) WVU 5F22 -

Salmonella Illinois (E3) ATCC 11646 -

Salmonella Senftenberg (E4) WVU 6F11 -

Salmonella Abaetetuba (F) ATCC 35640 -

Salmonella Poona (G1) DSM 109 -

Salmonella Cubana (G2) ATCC 12007 -

Salmonella Pomona (M) ATCC 10729 -

Bacillus subtilis ATCC 6633 -

Aeromonas veronii ATCC 51106 -

Citrobacter koseri ATCC 27026 -

Citrobacter freundii ATCC 8090 -

Enterobacter cloacae ATCC 27508 -

Enterobacter aerogenes ATCC 15038 -







Proteus vulgaris ATCC 8427 -

Proteus mirabilis ATCC 4630 -

Serratia liquefaciens ATCC 27592 -




13.10.1.1 Methodology 1 Salmonella Enteritidis ATCC 13076 and Salmonella Enteritidis ARS 2 11 single colony isolates were cultured in 10 mL of RapidChek 3

SELECTTM

primary enrichment media for 16-22 hr at 42 C. Then, 4 0.1 mL of the primary broth per mL of RapidChek SELECT

TM 5

secondary enrichment media required was transferred to secondary 6

enrichment broth. This was incubated at 42 C for 16-22 hr. The 7 samples were then diluted into RapidChek Select secondary media to 8 approximately 1 x 10

6 CFU/mL (approximately 1 log10 above the 9

detection limit of the method). Salmonella Kentucky ATCC 9263 10 was grown in non-selective media (TSB) (approximately 1 x 10

9 11

CFU/mL). Following enrichment, 1 mL aliquots of the enriched 12 sample was subjected to IMS as described in Section 13.1.1.2 while 13 varying the sample-IMS bead mixing time (10, 15, and 20 min). Five 14 (5) replicate aliquots were tested per bacterial strain per mixing time. 15 The samples (10 µL) were struck to BGN and XLT4 plates. The 16 plates were incubated at 35 ± 2°C for 18-24 h at which time 1 typical 17 colony was picked for confirmation. Typical colonies were 18

transferred to TSI and LSI slants and incubated at 35 C for 24 2 h. 19 The positive TSI slant was subjected to slide agglutination serology 20 testing for Salmonella Group D1. 21

22 13.10.1.2 Results 23 24

The results of this study are shown in Table 10. 25 26

Table 10. Results from the Immunomagnetic Separation (IMS) Method 27 Ruggedness Study- Incubation Time 28

29 30

The IMS confirmation procedure effectively detected SE at all sample 31 incubation times tested (10-20 min). 32

33

Test StrainTest Level,

CFU/mL10 15 20

Salmonella

Enteritidis

ARS 11

1 x 106 5/5 5/5 5/5

Salmonella

Enteritidis

ATCC 13076

1 x 106 5/5 5/5 5/5

Salmonella

Kentucky

ATCC 9263

1 x 109

(Neat) 0/5 0/5 0/5

No. Serogroup D1 Positives/ 5 Replicates

Incubation Time, min


13.10.2 Sample Volume 1 2

13.10.2.1 Methodology 3 4

Salmonella Enteritidis ATCC 13076 and Salmonella Enteritidis ARS 5 11 single colony isolates were cultured in 10 mL of RapidChek 6

SELECTTM

primary enrichment media for 16-22 hr at 42 C. Then, 7 0.1 mL of the primary broth per mL of RapidChek SELECT

TM 8

secondary enrichment media required was transferred to secondary 9

enrichment broth. This was incubated at 42 C for 16-22 hr. The 10 samples were then diluted into RapidChek Select secondary media to 11 approximately 1 x 10


detection limit of the method). Salmonella Kentucky ATCC 9263 13 was grown in non-selective media (TSB) (approximately 1 x 10

9 14

CFU/mL). Following enrichment, aliquots of the enriched sample 15 was subjected to IMS as described in Section 13.1.1.2 while varying 16 sample aliquot volumes (0.8, 1.0, and 1.2 mL). Five (5) replicate 17 aliquots were tested per bacterial strain per sample volume. The 18 samples (10 µL) were struck to BGN and XLT4 plates. The plates 19 were incubated at 35 ± 2°C for 18-24 h at which time 1 typical colony 20 was picked for confirmation. Typical colonies were transferred to 21

TSI and LSI slants and incubated at 35 C for 24 2 h. The positive 22 TSI slant was subjected to slide agglutination serology testing for 23 Salmonella Group D1. 24

25 13.10.2.2 Results 26 27

The results of this study are shown in Table 11. 28 29

Table 11. Results from the Immunomagnetic Separation (IMS) Method 30 Ruggedness Study- Sample Volume 31

32 33

Test StrainTest Level,

CFU/mL0.8 1 1.2

Salmonella

Enteritidis

ARS 11

1 x 106 5/5 5/5 5/5

Salmonella

Enteritidis

ATCC 13076

1 x 106 5/5 5/5 5/5

Salmonella

Kentucky

ATCC 9263

1 x 109

(Neat) 0/5 0/5 0/5


Sample Volume, mL


The IMS confirmation procedure effectively detected SE at all sample 1 volumes tested (0.8-1.2 mL). 2

3 13.11 Immunomagnetic Separation (IMS) Reagent Stability Study 4 5


Three lots of IMS reagents, aged approximately 2 months since 8 manufacturing, and stored at 4°C in final packaging (10 mL polypropylene 9 bottle), were placed into storage at various temperatures (4°C (recommended 10 storage condition), room temperature (20-22°C), 37°C (accelerated stability), 11 and 45°C (accelerated stability)). At 7 day intervals, an aliquot of each 12 reagent lot was removed from each storage condition and tested in replicate 13 (5) with samples of Salmonella Enteritidis ATCC 13076 and Salmonella 14 Enteritidis ARS 11 at 1 x 10


detection limit of the method) grown in the RapidChek Select media system 16 and Salmonella Kentucky ATCC 9263 grown in non-selective media (TSB) 17 and tested neat (approximately 1 x 10

9 CFU/mL). Following enrichment, 1 18

mL aliquots of the samples were subjected to IMS as described in Section 19 13.1.1.2. Five (5) replicate aliquots were tested per bacterial strain per 20 reagent lot per storage temperature. The samples (10 µL) were struck to 21 BGN and XLT4 plates. The plates were incubated at 35 ± 2°C for 18-24 h at 22 which time 1 typical colony was picked for confirmation. Typical colonies 23

were transferred to TSI and incubated at 35 C for 24 2 h. The positive TSI 24 slant was subjected to slide agglutination serology testing for Salmonella 25 Group D1. 26

27 13.11.2 Results 28 29

The results from this study are shown Table 12. 30 31 Table 12. Results of the Accelerated Immunomagnetic Separation (IMS) 32 Reagent Stability Study. 33

34 35

Time, weeks Test StrainTest Level,

CFU/mLLot 1 Lot 2 Lot 3 Lot 1 Lot 2 Lot 3 Lot 1 Lot 2 Lot 3 Lot 1 Lot 2 Lot 3

0 5/5 5/5 5/5 - - - - - - - - -

1 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5

2 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5

4 5/5 5/5 5/5 5/5 5/5 5/5

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

1 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5

2 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5

4 5/5 5/5 5/5 5/5 5/5 5/5

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

1 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5

2 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5

4 0/5 0/5 0/5 0/5 0/5 0/5

Salmonella

Enteritidis

ATCC

13076

1 x 106

Salmonella

Kentucky

ATCC 9263

1 x 109

(Neat)


4C Room Temperature 37C 45C

Salmonella

Enteritidis

ARS 11

1 x 106


The IMS reagent showed good stability for up to 2 weeks at 37 C and 45 C 1

and up to 4 weeks at 4 C to room temperature (20-25 C). This data supports 2

a shelf-life of up to 1 year at 4 C. 3 4 14. Independent Validation Studies 5

6 Validation studies conducted by the independent laboratory were under the direction of 7 the AOAC Research Institute. 8

9 14.1 Poultry House Drag Swab Method Comparison Study 10

11 Poultry house drag swabs were collected using standard procedures 12 (“Environmental Sampling and Detection of Salmonella in Poultry Houses” US 13 FDA, October 2008) from an egg layer facility with no history of SE 14 contamination. For spiking, a single colony isolate of Salmonella Enteritidis 15 ARS 12 (originally isolated from poultry house soil) was grown in non-selective 16 broth. Viable cell enumeration was accomplished by dilution plate counting on 17 non-selective agar plates. Individual drag swabs were inoculated at a target level 18 of 5 CFU/swab. Two (2) sets of 5 unspiked and 20 spiked drag swab samples 19

were acclimated at 4 C for 48 h, tested by either the SDIX method or the FDA 20 BAM reference method, and results compared. 21


Methods used for the Independent study were the same as those used for the 25 Internal study and are described in sections 14.1.1.1 and 14.1.1.2. The 26 Independent method comparison study used poultry house drag swab samples 27 collected at the same time as the Internal method comparison study. 28 29

14.1.2 Results 30 31 The results from the study are shown in Table 13. Eleven (11) samples were 32 positive with the RapidChek SELECT


presumptive positives samples confirmed as Salmonella Group D1 positive. 34 None of the test strip-negative samples (14) were culturally-positive. The 35 FDA-BAM reference method detected 6 positive samples. This gave an 36 accuracy of 183% indicating that 1.83 times more positives were found with 37 the RapidChek SELECT

TM SE method than with the reference method. The 38

chi-square value was 2.49, indicating the RapidChek method was statistically 39 equivalent to the reference method for the detection of SE in poultry house 40 drag swabs. 41


Table 13. Results from the Poultry House Drag Swab Method Comparison Study-Independent Laboratory Study. 1


bSensitivity Rate = (No. of test method presumptive positives)/(No. of test method confirmed positives) x 100.

cFalse 3

Negative Rate = 100 - Sensitivity Rate. dSpecificity Rate = (No. of test method negatives)/(No. of confirmed test method negatives) x 100.

eFalse Positive Rate 4

= 100 - Specificity Rate. fAccuracy = (No. of test method positives)/(No. of reference method positives) x 100. 5


Samples

Inoculation

Level,

CFU/sample

Presumptive

Positives

Confirmed

Positives

Reference

Method

Positives

Chi squarea

Sensitivity

Rateb

False

Negative

Ratec

Specificity

Rated

False Positive

Ratee Accuracy

f

5 0 0 0 0

20 5 11 11 6

100.0% 0.0% 183.0%

Poultry

House Drag

Swabs

S. Enteritidis

ARS 12

RapidChek

SELECT2.490 100.0% 0.0%


1 15. Discussion 2

3 The RapidChek SELECT

TM Salmonella Enteritidis Test Method was validated for the 4

detection of Salmonella Enteritidis (SE) in poultry house drag swab samples, shell egg 5 pools, and carcass rinsate samples. For the detection of SE in poultry house drag swab 6 samples, a immunomagnetic separation (IMS) method was used to aid in the isolation 7 and confirmation of SE from those samples. The test method showed equivalency to 8 both reference methods used for the detection of SE in poultry house drag swabs and 9 shell egg pools (FDA-BAM) as well as carcass rinsates (USDA-FSIS). The test method 10 gave a sensitivity of 100% and a specificity of 100% across all sample types. There 11 were no false positives or false negatives found in the study. The overall accuracy was 12 137%, indicating that, in general, the test method gave more positives (52) than the 13 reference methods (38). The overall Chi square was 4.95, indicating that the test method 14 was overall more sensitive than the reference method in this study. 15

16 The test method was highly selective for Salmonella Enteritidis and other Salmonella 17 Group D1 serotypes and did not cross-react with other commonly occurring bacteria 18 spanning 10 bacterial genera including several non-Group D1Salmonella. Both the 19 lateral flow test strip and the IMS reagent demonstrated very good accelerated stability 20 at elevated temperatures. 21 22

16. References 23 24

1. Mead, P.S., L., Slutsker, V. Dietz, F. McCraig, J.S. Bresee, C. Hapiro, P.M. Griffin, 25 and R.V. Tauxe (1999) Food-related illness and death in the United States. Emerg. 26 Infect. Dis. 5, 609-625. 27

28 2. Preliminary FoodNet data on the incidence of infection with pathogens transmitted 29

commonly through food- 10 states, 2009. MMWR Morb. Mortal. Wkly Rep. 2010 30 April 16; 59 (14), 418-422. 31 32

3. Altekruse, S.F., Bauer, N., DeSagun, R., Naugle, A., Schlosser, W., Umholtz, R., 33 White, P. Salmonella Enteritidis in broiler chickens, United States, 2000-2005. 34 Emerg. Infect. Dis. 12, 1848-1852. 35 36

4. Baker, A.R., Ebel, E.D., Hogue, A.T., McDowell, R.M., Morales, R.A., Schlosser, 37 W.D., Whiting, R. Salmonella Enteritidis Risk Assessment: Shell eggs and egg 38 products. United States Department of Agriculture, Washington, D.C. June 12, 1998. 39 40

5. US Department of Health and Human Services. Prevention of Salmonella Enteritidis 41 in shell eggs during production, storage, and transportation: Final Rule. Federal 42 Register 21 CFR Parts 16 and 118, July 9, 2009. 43

44 45


6. US Food and Drug Administration. Environmental Sampling and Detection of 1 Salmonella in Poultry Houses, October 2008. 2 http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/ucm114716.htm 3

4 7. US Food and Drug Administration, Center for Food Safety and Applied Nutrition. 5

Bacteriological Analytical Manual. Chapter 5: Salmonella. 6 http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnaly7 ticalManualBAM/default.htm. 8

9 8. US Department of Agriculture, Food Safety and Inspection Service. Microbiological 10

Laboratory Guidelines. Chapter 4: Isolation and identification of Salmonella from 11 meat, poultry and egg products. 12

http://www.fsis.usda.gov/Science/Microbiological_Lab_Guidebook/. 13 14

15

http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/ucm114716.htm

http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/default.htm

http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/default.htm

http://www.fsis.usda.gov/Science/Microbiological_Lab_Guidebook/

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