Microbiology Methods for ERP Review 3-2020

Microbiology Methods for Salmonella for AOAC ERP Review

AOAC INTERNATIONAL 2275 Research Blvd, Suite 300 Rockville, MD 20850

For questions on these reviews, please contact Deborah McKenzie at dmckenzie@aoac.org.

Microbiology Methods for Salmonella for AOAC ERP Review

METHOD REVIEW FORM URL: https://form.jotform.com/52106307924147

SAFETY REVIEW FORM URL: https://form.jotform.com/AOACINT/RISafetyRev

Table of Contents

3

Draft ERP Meeting Agenda (To Be Added)

4

OMA References and Resources

6

GENE ‐ UP Salmonella (SLM2) Collaborative Study Manuscript

52 53 54 75

‐ Safety Review (place holder) ‐ Statistics Review (place holder)

‐ Approved AOAC Collaborative Study Protocol

‐ PTM certification information

149

‐ User Manual/Package Inserts/IFU and MSDS Information

151 185 186 190 219 306

Solus One Salmonella Collaborative Study Manuscript

‐ Safety Review (place holder)

‐ Statistics Review

‐ Approved AOAC Collaborative Study Protocol

‐ PTM certification information

‐ User Manual/Package Inserts/IFU and MSDS Information

3

ERP MEETING AGNDA - TO BE ADDED

4

OMA PROGRAM – GUIDELINES & REFERENCES

Please note that the following references are website links listed for your use and reference during the protocol development and manuscript/method review process. These appendices, guidelines, and references can be accessed via the AOAC website.

These are website links, please press “CTRL” and select the document.

MICROBIOLOGY METHODS

A. Appendix J: AOAC International Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces

CHEMISTRY METHODS /OTHER

B. Appendix A: Standard Solutions and Reference Materials C. Appendix B: Laboratory Safety D. Appendix C: Reference Tables E. Appendix D: Guidelines for Collaborative Study Procedures to Validate Characteristics of a Method of Analysis F. Appendix F: Guidelines for Standard Method Performance Requirements G. Appendix G: Procedures and Guidelines for the Use of AOAC Voluntary Consensus Standards to Evaluate Characteristics of a Method of Analysis H. Appendix H: Probability of Detection (POD) as a Statistical Model for The Validation of Qualitative Methods I. Appendix I: AOAC International Methods Committee Guidelines for Validation of Biological Threat Agent Methods and/or Procedures J. Appendix K: Guidelines for Dietary Supplements and Botanicals K. Appendix L: AOAC Recommended Guidelines for Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) Single-Laboratory Validation L. Appendix M: Validation Procedures for Quantitative Food Allergen Elisa Methods: Community Guidance and Best Practices M. Appendix N: ISPAM Guidelines for Validation of Qualitative Binary Chemistry Methods N. Standard Method Performance Requirements (SMPR) Guidelines (Appendix F) O. Official Methods Board Expert Review Panels (ERP) Guidelines (Appendix G) P. How to Write in AOAC (OMA) Style A. Dietary Supplements SLV Guidelines (Appendix K) B. Food Microbiology Guidelines (Appendix J) C. Stakeholder Panel on Infant Formula & Adult Nutritionals (SPIFAN) SLV Guidelines (Appendix L) A. Food Microbiology Guidelines (Appendix J) B. Collaborative Study Validation Guidelines (Appendix D) C. Biological Threat Agent Methods (BTAM) (Appendix I) D. Characterization of Antibodies Used in Immunochemical Methods of Analysis for Mycotoxins and Phycotoxins A. AOAC Interlaboratory Study Workbook - Blind (Unpaired) Replicates (Excel file .xls) B. AOAC Interlaboratory Study Workbook - Binary Data (Excel file .xls) C. LCF MPN Calculator - Ver. 1.6 (Zip file .zip)

GUIDELINES

SINGLE-LAB GUIDELINES & REFERENCES

MULTI-LAB GUIDELINES & REFERENCES

S TATISTICAL/MATHEMATICAL TOOLS

www.aoac.org

5

OMA PROGRAM – GUIDELINES & REFERENCES

TECHNICAL BULLETINS The documents posted in this tab are not a replacement of validation guidelines or any standard method performance requirements. These documents are not AOAC policy or procedure. These documents summarize the decisions of the AOAC Expert Review Panel(s) (ERPs) formerly administered by the AOAC Research Institute related to method approval that provide clarification and that may be applicable to the development of validation study designs. A. Various clarifications and recommendations of the AOAC ERP for Microbiology Methods for Foods and Environmental Surfaces (May 15, 2016) B. Acceptable Multi-matrix Validation Claims for Food Microbiology Methods (May 15, 2016)

OTHER REFERENCE DOCUMENTS

A. First Action Official Methods SM of Analysis _Flowchart B. First Action Official Methods SM of Analysis Requirements

C. AOAC First Action Methods Orientation D. AOAC Final Action Methods Orientation

www.aoac.org

6

Evaluation of the GENE-UP ® Salmonella Method for the Detection of Salmonella Species in a Broad Range of Foods and Select Environmental Surfaces: Collaborative Study

Ronald Johnson 1 , John Mills & Nikki Taylor

bioMérieux, Inc. 595 Anglum Road, Hazelwood, MO, 63042 USA Patrick M. Bird AOAC INTERNATIONAL, 2275 Research Blvd. #300, Rockville, MD, 20850 USA

Background : The GENE-UP ® Salmonella (SLM) assay ( Performance Tested Method SM 121802) is a PCR detection method that utilizes Fluorescence Resonance Energy Transfer hybridization probes for the rapid detection of Salmonella species in foods and on environmental surfaces. Objective : The purpose of this validation was to evaluate the method’s interlaboratory performance for submission to AOAC INTERNATIONAL for adoption as First Action Official Method of Analysis SM . Method : The GENE-UP ® method was evaluated in a multi-laboratory study using unpaired test portions for one food matrix, raw ground beef (80% lean). The candidate method was compared to the USDA FSIS MLG 4.10 reference method. An alternative confirmation procedure, using ASAP™ and CHROMID ® Salmonella chromogenic agars, was included in the validation study. Fifteen collaborators from 14 laboratories throughout the

1 Corresponding author. Ron.Johnson@biomerieux.com

1

7

United States participated. Three levels of contamination were evaluated; a non- inoculated control level (0 CFU/test portion) , a low contamination level (~0.7 CFU/test portion) and a high contamination level (~2 CFU/test portion). Data were analyzed using the Probability of Detection (POD) statistical model as presented in the AOAC validation guidelines. Results : The dLPOD C values with 95% confidence interval for the GENE-UP Salmonella method with either alternative or traditional confirmation were: 0.00 (-0.03, 0.03), -0.02 (-0.15, 0.12) and 0.02 (-0.03, 0.09) for the non-inoculated, low and high contamination levels respectively. Conclusion : The dLPOD C results demonstrate no difference in performance between the candidate method and reference method for the matrix evaluated. Highlights: The GENE-UP Salmonella method, with ASAP and CHROMID Salmonella , provides industry with a simplified, rapid and accurate workflow for the detection of Salmonella in a broad range of foods and select environmental surfaces. _________________________________________________________________________ Introduction Salmonella, one of the leading causes of foodborne illness, has been a known causative agent of outbreaks for over 100 years. In the United States, it is estimated that each year more than 1 million people fall ill to the bacterium, with approximately 20,000 hospitalizations and nearly 400 deaths (1). The Center for Disease Control and Prevention (CDC, Atlanta, GA) notes that Salmonella continues to have one of the two highest incidence of infections per 100,000 people (2). The impact from these illnesses has resulted in an estimated $3 billion cost to our healthcare system (3). The persistence of this organisms in our food chain underscores the need for reliable and accurate methods of detection. The GENE-UP ® Salmonella (SLM) utilizes a unique PCR technology that employs a dual

2

8

hybridization probe approach to detect Salmonella in a broad range of foods and environmental samples. The method combines the detection of real-time amplification curves and melt peaks to determine positive or negative results. Presumptive positive results can be obtained in as little as 12–26 h, depending on the matrix, and confirmation can be initiated directly from the primary enrichment by streaking onto ASAP™ and/or CHROMID® Salmonella. Prior to the collaborative study, the GENE-UP ® method was validated according to the AOAC Guidelines (4) in a Performance Tested Method SM (PTM) study. The method was originally validated for liquid whole egg (25 mL), cooked ham (25 g), raw ground beef (25 g), raw chicken (25 g), lettuce (25 g), chocolate (25 g), dry pet food (25 g) and stainless-steel environmental surface sponges. It was awarded PTM Certification No. 030803 in March of 2008. Several modification studies were conducted to extend the matrix claims of the assay. In June of 2015, the method was validated for fresh raw ground beef (80% lean, 25 g & 375 g), fresh raw chicken breast (25 g), vanilla ice cream (25 g), dry pet food (25 g) and stainless-steel environmental sponges and awarded PTM Certification No. 061404. In July of 2017, the method was validated for chicken carcass rinse (30 mL), raw ground chicken (25 g), raw beef trim (375 g), raw ground pork (375 g), raw ground bison (375 g), pasteurized liquid egg (100 g), powdered egg (100 g), whey protein powder (375 g), dry pet food (375 g), creamy peanut butter (375 g), dark chocolate (375 g) and garlic powder (375 g). The method was further validated in December 2017 for bulk bagged Romaine lettuce (375 g), and again for dark chocolate (375 g, 42 o C enrichment temperature) in December 2018 and a new certification number, PTM Certification No. 121802, was awarded (5) . Two additional matrices, milk chocolate (25 g & 375 g) and dark chocolate (375 g; 36 o C enrichment temperature) were submitted for PTM certification in January of 2020. During the PTM studies, the candidate method was compared to the USDA – Food Safety Inspection Service (FSIS) Microbiology Laboratory Guidebook (MLG) Salmonella Chapter (6)

3

9

and the US Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Chapter 5 Salmonella (7) reference methods. The method comparison studies were conducted using the current versions of the reference methods available at the time of validation. PTM studies included product consistency and stability studies, robustness studies, inclusivity and exclusivity studies and an independent laboratory evaluation. The purpose of this collaborative study was to meet the requirements for submission as an AOAC Official Methods of Analysis SM based on current AOAC guidelines. The study compared the reproducibility of the candidate method for one matrix, raw ground beef (80% lean), using a composite test portion, to the USDA FSIS MLG 4.10 reference method.

Collaborative Study

Study Design

One matrix, raw ground beef (80% lean), was evaluated in the study. The matrix was obtained from a local butcher and screened for the presence of Salmonella by the GENE-UP SLM and MLG 4.10 reference methods. The matrix was artificially contaminated with S. Typhimurium NCTC 12023 (Bovine, septicemic liver) at two levels of contamination: a high inoculation level of ~2 CFU/test portion and a low inoculation level of ~0.7 CFU/test portion. A set of non-inoculated control test portions (0 CFU/test portion) were also included. Twelve replicate samples from each of the three inoculation levels were analyzed by each participant. A total of 72 unpaired samples, 36 for the candidate method and 36 for the MLG 4.10 reference method, was sent to each participant. Collaborators were also sent a test portion for determining the total aerobic plate count (APC) using the FDA BAM Chapter 3 reference method (8) or TEMPO ® AC (9).

4

10

Preparation of Inoculum and Test Portions

The Salmonella strain used in this evaluation was rehydrated from lyophilized pellets (BIOBALL ® , bioMérieux, #413775) into 10 mL buffered peptone water. Once rehydrated, the culture was homogenized thoroughly and dilutions (1:10) were prepared in Butterfield’s phosphate buffered diluent (BPD) for both the low and high inoculation levels. A bulk lot of matrix was inoculated with 10 mL of culture for each contamination level, homogenized by hand mixing and packaged into individual test portions for the GENE-UP SLM method or reference method. For the GENE-UP SLM test portions, 25 g of inoculated test material was combined with 350 g of non-inoculated test material. Reference method test portions were 25 g.

Test Portion Distribution

Samples were blind-coded and shipped to the collaborating sites at refrigerated temperature (2-8°C). Samples were received on a Friday and held at 2-8 o C until analysis was initiated the following Monday. Within each shipment, a sample labeled temperature control was included. Participants were instructed to report the temperature of the samples on the sample receipt form and return to the study directors. Temperature records for each laboratory have been included with the supplementary materials.

Test Portion Analysis

Test portions (375 g) evaluated by the candidate method were enriched with 1125 mL of BPW (prewarmed to 42 ± 1 o C), homogenized for 2 min and incubated for 10 h at 42 ± 1°C.

5

11

Following incubation, a 20 μ L aliquot of each sample was lysed for 5 min at >2000 rpm. Lysed samples were transferred to PCR reaction tubes and assayed on the GENE-UP ® thermocycler. Regardless of presumptive results, all test portions were confirmed following two procedures: a traditional confirmation following the MLG 4.10 reference method beginning with a transfer to secondary enrichments and an alternative confirmation procedure by directly streaking the primary enrichment broth to ASAP and CHROMID Salmonella and incubating at 35 ± 1°C for 24 ± 2 h. Isolated colonies from both the traditional and alternative confirmation procedures were confirmed following the procedures outline in MLG 4.10 with biochemical confirmation conducted using VITEK 2 GN (OMA 2011.17) or API 20E (OMA 978.24). For test portions evaluated by the reference method, 25 g samples were enriched in 75 mL mTSB and incubated for 15–24 h at 42 ± 1°C. After incubation, an aliquot of the mTSB was transferred to secondary enrichments (Tetrathionate broth, Hajna, TT and Rappaport-Vassiliadis medium with soya, RVS). Secondary enrichment tubes were incubated at 42 ± 0.5 o C for 22-24 h in an incubator or for 18-24 h in a circulating water bath. After incubation, secondary enrichments were struck to selective agars (Brilliant green sulfa agar, BGS and double modified lysine iron agar, DMLIA) and incubated at 35 ± 1°C for 18-24 h. Regardless of the presence of typical colonies, all plates were reincubated for an additional 18-24 h. Isolated colonies from the selective agars were transferred to triple sugar iron agar (TSI) and lysine iron agar (LIA) slants and incubated at 35 ± 1°C for 24 ± 2 h. Final biochemical confirmation was conducted using VITEK 2 GN or API 20E.

Statistical Analysis

Data for each contamination level was analyzed using the probability of detection (POD) statistical model (10) and conducted using the AOAC Micro Stats Workbook V1.0. The POD

6

was calculated as the number of positive outcomes divided by the total number of trials. Laboratory POD (LPOD) values were calculated as the total POD values for all collaborators. The LPOD was calculated for the candidate presumptive results, LPOD CP, the candidate confirmatory results (including false negative results), LPOD CC , the difference in the candidate presumptive and confirmatory results, dLPOD CP, presumptive candidate results that confirmed positive (excluding false negative results), LPOD C, the reference method, LPOD R , and the difference in the confirmed candidate and reference methods, dLPOD C . A dLPOD C confidence interval not containing the point zero would indicate a statistically significant difference between the candidate method and the reference method at the 95 % confidence level. In addition to POD values, the repeatability standard deviation (s r ), the among laboratory repeatability standard deviation (s L ), the reproducibility standard deviation (s R ) and the standard error were calculated. 12

7

13

AOAC Official Method 2020.xxx Salmonella species in a Broad Range of Foods and Select Environmental Samples: GENE-UP ® Salmonella (SLM) Test Method First Action 2020 Applicable to detection of Salmonella in 25 g test portions of fresh raw ground chicken, fresh raw chicken breast, fresh raw ground beef (80% lean), fresh raw fish, creamy peanut butter, vanilla ice cream, dry pet food and milk chocolate; for 30 mL test portions of chicken carcass rinse; for 100 g test portions of pasteurized liquid eggs and powdered egg; for 375 g test portions of fresh raw ground beef (80% lean), raw beef trim, raw ground pork, raw ground bison, whey protein powder, dry pet food, creamy peanut butter, garlic powder, bulk bagged Romaine lettuce, dark chocolate and milk chocolate; for stainless steel environmental sponges.

See Table 2020.1A-B for a summary of results of the inter-laboratory study. See Table 2020.2A-B for detailed results of the inter-laboratory study

A. Principle

The GENE-UP ® Salmonella (SLM) kit is designed for use with the GENE-UP ® Thermocycler. The GENE-UP ® SLM kit contains all of the necessary components for PCR, including sample-specific primers and probes and an internal amplification control. The GENE- UP ® Thermocycler detects fluorescence at several wavelengths (channels) to allow for multi- target detection in the same reaction vessel. The fluorescent signal from a sample is recorded in channel 640, while the fluorescent signal for an internal amplification control is recorded in channel 705. The software automatically interprets the results for the internal amplification

8

14

control and determines the sample result based on the outcome of the control. Both the assay for the sample and in the internal amplification control utilize dual Fluorescence Resonance Energy Transfer (FRET) hybridization probes. These probes consist of two different short oligonucleotides that hybridize to an internal sequence of the amplified fragment during the annealing phase of the reaction cycle. The first probe for the same assay is labeled at the 3’ end with fluorescein; the second probe is labeled at the 5’ end with LC Red 640. FRET occurs only after the two probes come in close proximity from hybridizing to the template DNA. The resulting fluorescent signal from the FRET interaction, which forms a real-time amplification curve, is how the amplified target is detected by the GENE-UP ® Thermocycler. After the PCR cycling program finishes, the PCR product(s) are melted to determine the presence of the target DNA. The software interprets data for each sample and gives a positive, negative, or inhibited result.

B. Apparatus and Reagents

Items available from bio Mérieux, Inc. (595 Anglum Road, Hazelwood, MO, 63042 USA) (a) GENE-UP ® Salmonella (SLM) . ― bioMérieux, Inc., REF 423105 (192 tests). Store kit at 2-25°C. (1) PCR Reagent Strips . ―Two pouches with 12 strips of 8 tubes (2 x 96 tubes). (2) Optical Caps . ―Two pouches with 12 strips of 8 tubes (2 x 96 caps).

(3) Control Buffer . ―One pouch with control buffer vial. (b) GENE-UP ® Lysis Kit. ―bioMérieux, Inc., REF 414057. (c) GENE-UP ® Thermocycler. ―bioMérieux, Inc., REF 414056. (d) Laboratory paddle blender for sample homogenization. (1) SMASHER ® . ― bioMérieux, Inc., REF AESAP1064.

9

15

(2) SMASHER ® XL . ― bioMérieux, Inc., REF AESAP1100. (e) Buffered Peptone Water . (1) 6 x 225 mL. ―bioMérieux, Inc., REF 42043. (2) 6 x 90 mL. ―bioMérieux, Inc., REF 42042, 42729. (3) 6 x 225 mL mini bag. ―bioMérieux, Inc., REF 42729. (4) 4 x 3 L. ―bioMérieux, Inc., REF AEB910303/4. (5) 2 x 5 L. ―bioMérieux, Inc., REF AEB910305/2. (f) Novobiocin. ―bioMérieux, Inc., REF AEB184150/10. (g) Advanced Digital Heavy. ―Duty Vortex Mixer. (1) 120 V. ―bioMérieux, Inc., REF 9456TAHDUSA. (2) 230 V. ―bioMérieux, Inc., REF 9456TAHDEUA. (h) GENE-UP ® Lysis Rack Adaptor. ―bioMérieux, Inc., REF 414570. (i) Sterile blender bags. (1) Type P filter bag. ―bioMérieux, Inc., REF AES400P/50G. (2) SMASHER ® XL Bag 2L. ―bioMérieux, Inc., REF 415180. (3) SMASHER ® XL Bag 2L. ―bioMérieux, Inc., REF 415181. (j) Plate Centrifuge. (1) MPS1000 Mini PCR Plate Spinner 110V. ―bioMérieux, Inc., REF 419196. (2) MPS1000 Mini PCR Plate Spinner 230V. ―bioMérieux, Inc., REF 414555. (k) GENE-UP ® PCR Tube Holder. ―bioMérieux, Inc., REF 414573. (l) GENE-UP ® Lysis Tube Remover. ―bioMérieux, Inc., REF 414469. (m) GENE-UP ® Heavy Rack Holder. ―bioMérieux, Inc., REF 414571. (n) GENE-UP ® Lysis Tube Holder. ―bioMérieux, Inc., REF 414572. (o) Troemner Vortex Mixer Adaptor. ―bioMérieux, Inc., REF 421713. (p) 10 µL Biotix filter pipette tip. ―bioMérieux, Inc., REF 419194.

10

16

Confirmation Materials (a) SALSA ™ Agar. ―bioMérieux, Inc., REF AEB125980/AEB526760. (b) XLD Agar. ―bioMérieux, Inc., REF 43563. (c) ASAP ™ Agar. ―bioMérieux, Inc., REF AEB520089/AEB520090. (d) CHROMID Salmonella. ―bioMérieux, Inc., REF 43621. (e) SX2 Broth. ―bioMérieux, Inc., REF 42121. (f) API ® 20E. ―bioMérieux, Inc., REF 20100. (g) Salmonella spp Latex kit. ―bioMérieux, Inc., REF MGNF42.

(h) VIDAS ® SPT. ―bioMérieux, Inc., REF 30707. (i) VIDAS ® SLM. ―bioMérieux, Inc., REF 30702.

Additional Items (b) Incubators . ― Capable of maintaining 41.5 ± 1ºC.

(c) Adjustable, variable volume pipettes . ―C apable of sampling and delivering 10–20 µL. (d) Adjustable, variable volume pipettes (single or multichannel) . ― Capable of sampling and delivering 0.5–10 µL.

(e) Compatible sterile, filter pipette tips for 20 µL . (f) Refrigerator . ― Capable of maintaining 2–8°C. (g) Vortex-Genie Pulse . ― Scientific Industries, SKU: SI-P236.

C. General Instructions

(a) The GENE-UP ® Salmonella (SLM) kit is for professional use only with the GENE-UP ® Lysis kit.

11

17

(b) Comply with Good Laboratory Practices. (c) Do not use reagents after the expiration date indicated on the label. (d) Visually inspect vials before testing. Do not use vials with evidence of damage, leakage, or deterioration. (e) Do not mix reagents (or disposables) from different lots. (f) Powder-free latex or nitrile gloves are recommended for all PCR steps. (g) The equipment and accessories should be regularly cleaned and decontaminated. (h) Never remove the caps from the lysis tubes. (i) Do not try to remove the strip tube caps once they have been sealed to the PCR strip tubes (j) Spills should be wiped up thoroughly after treatment with bleach or a nucleic acid degradation solution. See the GENE-UP ® user manual for information on cleaning spills on or in the instrument. Do not autoclave solutions containing bleach. Dispose of used or unused reagents as well as any other contaminated disposable materials following procedures for infectious or potentially infectious products. It is the responsibility of each laboratory to handle waste and effluents produced according to their type and degree of hazardousness and to treat and dispose of them (or have them treated and disposed of) in accordance with any applicable regulations. Salmonella is a Gram-negative facultative rod-shaped bacterium organism. Care must be taken when handling samples that may contain Salmonella . Strict compliance with BSL-2 practices, containment equipment, and facilities are recommended for all activities utilizing known or potentially infectious clinical materials or Safety Precautions

12

18

cultures. While BSL-2 containment is suitable for all other salmonellae, BSL-3 practices and equipment are recommended for activities likely to produce significant aerosols or for activities involving production quantities of this particular organism. Laboratory personnel must be adequately trained to handle pathogens before being permitted to analyze samples for Salmonella . Follow appropriate safety guidelines when handling potentially contaminated samples. Waste should be disposed of in compliance with local and national legislation. S. enterica serovar Typhi ( Salmonella Typhi) is the causative agent of typhoid fever and is a documented hazard to laboratory personnel as Salmonella may be present in feces, blood and urine. Laboratory-acquired Salmonella Typhi infections usually present with symptoms of septicemia, headache, abdominal pain and high fever, and it causes death more than other salmonellae. The infectious dose is low (<10 3 organisms), and the incubation period may vary from one to six weeks, depending upon the dose of the organism. Vaccines for Salmonella Typhi are available and should be considered for personnel regularly working with potentially infectious materials.

D. Sample Enrichment

Allow the enrichment broths to reach 15–25°C before use. In some cases, the enrichment media should be pre-warmed to 42 ± 1°C before adding to food samples. Frozen samples should be thawed before analysis. Use a blender bag containing filter.

(a) Fresh raw ground chicken, fresh raw ground beef, fresh raw chicken breast, fresh raw fish, creamy peanut butter, vanilla ice cream and dry pet food (25 g) . ― Add 225 mL BPW. Homogenize and incubate at 42 ± 1°C for 18–24 h.

13

19

(b) Milk chocolate (25 g) . ― Add 225 mL of reconstituted non-fat dry milk. Homogenize and incubate at 35 ± 1°C for 22-26h. (c) Chicken carcass rinse (30 mL) . ― Add 30 mL of pre-warmed (42 ± 1°C) BPW. Homogenize and incubate at 42 ± 1°C for 18–24 h. (d) Raw beef trim, raw ground pork and raw ground bison (375 g) . ― Add 1125 mL of pre- warmed (42 ± 1°C) BPW or modified tryptic soy broth (mTSB). Homogenize and incubate at 42 ± 1°C for 10–24 h. Note – only BPW can be used for raw ground bison. (h) Romaine lettuce (375 g) . ― Add 1125 mL of pre-warmed (42 ± 1°C) BPW. Homogenize and incubate at 42 ± 1°C for 22–26 h. (1) Five enriched samples can also be wet pooled. After incubation, mix the contents of each sample bag and combine 1 mL from each sample into a prelabeled tube. (i) Pasteurized liquid egg and powdered egg (100 g) . ― Add 900 mL of pre-warmed (42 ± 1°C) BPW. Homogenize and incubate at 42 ± 1°C for 22–26 h. (j) Whey protein powder & instant non-fat dried milk (375 g) . ― Add 1125 mL of pre- warmed (42 ± 1°C) BPW. Homogenize and incubate at 42 ± 1°C for 22–26 h. (k) Garlic powder (375 g) . ― Add 3375 mL of pre-warmed (42 ± 1°C) BPW + K 2 SO 3 . Homogenize and incubate at 42 ± 1°C for 22–26 h. (l) Creamy peanut butter (375 g) . ― Add 3375 mL of pre-warmed (42 ± 1°C) BPW. Homogenize and incubate at 42 ± 1°C for 20–24 h. (m) Dark chocolate (375 g) . ― Add 3375 mL of pre-warmed (42 ± 1°C) BPW. Homogenize and incubate at 42 ± 1°C for 10–24 h. Alternatively, add 3375 mL of pre-warmed (36 ± 1°C) BPW. Homogenize and incubate at 36 ± 1°C for 22-26h. (n) Milk Chocolate (375g) . ― Add 3375 mL of reconstituted non-fat dry milk containing 0.01% brilliant green solution. Homogenize and incubate at 35 ± 1°C for 22-26h. (o) Dry pet food (375 g) . ― Add 3375 mL of pre-warmed (42 ± 1°C) BPW. Homogenize

14

20

and incubate at 42 ± 1°C for 22–26 h. (p) Environmental samples . ― Add enough BPW to cover the sampling device (ex. 10 mL for a swab and 100 mL for a sponge). Homogenize and incubate at 42 ± 1°C for 18–24 h. Note: For environmental samples, the collection device should first be dampened with a sterile diluent (e.g., buffered peptone water) containing, if necessary, a suitable neutralizing agent (e.g., Lecithin‑Polysorbate‑L-Histidine‑Sodium thiosulfate mixture or Dey Engley). Note: Incubation conditions may have repercussions on short detection procedures. The temperatures indicated must be scrupulously respected. In particular, it is advisable to ensure that the conditions for preheating the enrichment broth enable the indicated temperature to be reached. The sample preparation time (time between the end of the enrichment broth pre‑heating phase and the start of the food sample incubation phase), must not exceed 45 minutes. It is recommended to use a ventilated incubator for the incubation phase. Note: Extending the enrichment time to 24 hours allows for performance improvement of the alternative method only for large portions of raw meat products . (a) Following incubation, manually mix the contents of the blender bag. Optionally, a sterile technique can be used to remove 1 mL of enriched sample; place it in a pre-labeled microcentrifuge tube. Note: Do not discard the individual enriched samples until the analysis is complete and it has been confirmed that no further testing is required. Enriched samples can be stored at 2–8°C for up to 72 h before performing analysis. (b) Use the plate map created in the GENE-UP ® Routine software to determine the number of lysis tubes required from the GENE-UP ® Lysis Kit and place the correct number of lysis tubes E. Sample Lysis

15

21

in the GENE-UP ® Lysis Tube Holder. If less than 8 tubes in a strip are required, the strips can be cut apart. Note: Never open the lysis tubes. If a lysis tube opens or leaks, this should be considered a contamination event. (c) Clip the GENE‑UP ® Lysis Tube Holder on the GENE‑UP ® Heavy Rack Holder. (d) Transfer 20 μL of sample into the lysis tube. Use the Plate Map from the GENE‑UP ® Routine software to pipet each sample into the correct plate position. (e) Remove the GENE‑UP ® Lysis Tube Holder from the GENE‑UP ® Heavy Rack Holder. (f) Clip the GENE‑UP ® Lysis Tube Holder on the Troemner Vortex Mixer Adaptor. (g) Run the vortex mixer at 2200 rpm for 5 min. Note: When using the Vortex‑Genie ® Pulse, fit it with the GENE‑UP ® Lysis Rack Adaptor. Run the vortex at maximum speed for 5 min. The maximum speed must be above 2000 rpm. (h) When lysis is complete, remove the GENE‑UP ® Lysis Tube Holder from the Troemner Vortex Mixer Adaptor. (i) Clip the GENE‑UP ® Lysis Tube Holder into the GENE‑UP ® Heavy Rack Holder and proceed to Final Setup for PCR. Note: The lysate can be stored for up to 3 days at 2–8°C or at -15 to -31°C for extended storage.

F. PCR Preparation

Before beginning the procedure, don a clean pair of powder-free latex or nitrile gloves. (a) Use the plate map created in the GENE‑UP ® Routine software to determine the number of PCR tubes required from the GENE‑UP ® PCR Kit and place the correct number of PCR tubes in the GENE‑UP ® PCR Tube Holder. If less than eight tubes in a strip are required, the strips can

16

22

be cut apart, and only the used tubes are placed in the GENE‑UP ® PCR Tube Holder. Note: Only remove the required number of strips from the pouch and carefully reseal the pouch after opening. (b) Use the following steps to remove the transportation caps from the strips: (1) Tap on the strips on the bench to ensure the pellets are on the bottom of the tubes. (2) Carefully open the caps to prevent spilling the freeze‑dried pellet. (3) Visually check that the freeze‑dried pellets are present at the bottom of each tube. (c) Using a 10 μL Biotix filter pipette tip with a single or multichannel pipette, transfer 10 μL of lysed sample (red) in the appropriate PCR tube. To determine the appropriate plate position for each sample, refer to the Plate Map from the GENE‑UP ® Routine software. Note: Do NOT agitate the lysate before aspirating the sample. The solid material must stay at the bottom of the tube. Note: Visually check the tips to confirm the absence of beads, bubbles, and for correct volume of lysate. Note: For negative control procedure, use 10 μL of control buffer instead of lysed sample. (d) Place and seal the strip caps onto each strip tube using the GENE‑UP ® Lysis Tube Remover Tool. If less than eight caps are required, the caps can be cut apart, and only the used caps are placed onto the strip tubes. (e) Place the GENE‑UP ® PCR Tube Holder containing the PCR tubes in the plate centrifuge. (f) Balance the centrifuge. (g) Spin for 10 s. (h) The plate is now ready to be processed in the GENE‑UP ® instrument and is stable for 2 h at 15–25°C. Note: The lysis tubes can be removed from the GENE‑UP ® Lysis Tube Holder using the

17

23

GENE‑UP ® Lysis Tube Remover Tool. The GENE‑UP ® Lysis Tube Holder is reusable, but the used lysis tubes should be disposed of accordingly. (i) Please refer to the appropriate GENE‑UP ® instrument user manual for instructions to start a run, view results, and use the GENE‑UP ® Routine software.

G. Results and Interpretation

(a) Results are automatically interpreted once the PCR run is completed. The routine software interprets data for each sample and gives a positive, negative, or inhibited result as indicated in the following table

Salmonella spp. (640 nm)

Internal amplification Control (705 nm)

Result

+ +

+

+ +

-

- -

+

-

! Inhibition

-

H. PCR Inhibition Protocol (a) In case of an inhibited result, dilute the lysate to 1:3 in the control buffer: (b) Transfer 10 μL of control buffer in an adapted microtube. (c) Follow the same procedure in the PCR PREPARATION section of this document, using 10 μL of this dilution of lysate. Note: It is recommended to retest in parallel the lysate without dilution. Note: In case of inhibited results at 1:3, you can dilute the lysate to 1:10. Note: Some matrices like aromatics herbs or cocoa powders may contain inhibitory molecules. For these matrices, 1 mL of enriched sample could be diluted 1:5 or 1:10 in Tryptone

18

24

Salt or Normal Saline prior to the lysis step. If inhibition persists, proceed with an additional 1:3 dilution as described above.

I. Confirmation of Positive Results

All positive results must be confirmed according to the BAM, MLG, or ISO (11) reference methods or according to the following bioMérieux GENE‑UP ® confirmation protocol. Confirmation should be performed using the enrichment broth stored at 2–8°C and should be initiated within 72 h following the end of the incubation period. If using an enriched sample, mix thoroughly by hand. Note: For short protocols, the confirmation can be made at the end of the incubation time (8 h or 10 h). (a) Isolate the enrichment broth on a SALSA ™ agar plate, CHROMID Salmonella or on ASAP and XLD agar plates; incubate at 35°C ± 1°C for 24 ± 3 h. (b) If a typical colony is obtained, test an isolated colony directly using a Salmonella spp. latex or API ® 20E, VITEK GN or VITEK MS.. In the event of discordant results (positive with the alternative method, not confirmed by one of the options described above), the laboratory must take the necessary steps to ensure that the results obtained are accurate. It is recommended, for example, to perform the following procedure:

(a) Transfer 100 μL from the enrichment to 10 mL SX2 for a second enrichment. Incubate at 42°C ± 1°C for 24 ± 3 h.

19

25

(b) Use a loop to isolate the sample directly from the second enrichment. (c) Streak the sample on SALSA, ASAP, CHROMID Salmonella and XLD agar plates. (d) Incubate at 35°C ± 1°C for 24 ± 3 h. (e) Alternatively, VIDAS ® SLM (or SPT) can be performed with an input volume of 500 μL of SX2 broth. (f) If no typical Salmonella colony is identified, the result is considered negative. (g) If a typical colony is obtained, test an isolated colony directly using a Salmonella spp. latex, API 20E strip, VITEK GN or VITEK MS. External quality control can be performed using one Salmonella strain. (a) Add one isolated colony from a fresh and pure culture in 9 mL of Buffer Peptone Water. (b) Mix and incubate at 42 ± 1°C for 18‑24 h. (c) Dilute 1/100 of the culture in BPW in order to obtain a suspension containing approximately 10 6 cells/mL of the strain. (d) Follow the protocol from the SAMPLE LYSIS section steps to CONFIRMATION OF POSITIVE RESULTS sections. (e) Check that the results obtained correspond to the characteristics of the tested strains. J. Quality Control

K. Limitations of the Method

The GENE‑UP ® Salmonella (SLM) kit has been evaluated on a large number of matrices. However, given the wide variety of products and manufacturing procedures, it is recommended to check that the composition of the matrices tested does not affect the reliability of GENE‑UP ®

20

26

results.

Results of Collaborative Study

The collaborative study involved a method comparison evaluation of the candidate method to the MLG 4.10 reference method for raw ground beef (80% lean). A total of 15 collaborators from 14 sites throughout the United States participated in this study. Each participant analyzed 36 unpaired test portions for the candidate method and the MLG reference method: 12 inoculated with a high level of Salmonella , 12 inoculated with a low level of Salmonella , and 12 non- inoculated controls. Of the 15 collaborators, 11 were included in the final statistical analysis. One laboratory was not able to complete testing and did not submit results. One laboratory did not perform the traditional confirmation procedure and were therefore excluded from the statistical analysis. Two laboratories (1 and 10) recovered the target analyte in their non- inoculated control samples and their data was not included in the statistical analysis. Tables 2020.1A and 1B summarize the results and POD statistical analysis of the collaborative study. Detailed results for each collaborator are presented in Table 2020.2A and 2020.2B. The individual collaborator test portion results are presented in Tables 1 and 2 of the Supplementary Materials. The concentration of Salmonella , and 95% confidence interval, in each inoculation level was determined on the day of initiation of analysis by the coordinating laboratory: 0.49 CFU/test portion (0.37, 0.63) for the low inoculum level and 3.43 CFU/test portion (2.72, 4.88) for the high inoculum level. The results of the APC analysis produced a range of 8.1 x 10 5 to 2.5 x 10 7 CFU/g for an average of 6.6 x 10 6 CFU/g. Complete APC results are presented in Table 3 of the Supplementary Materials. Temperature records obtained during the shipment of samples are presented in Table 4 of the Supplementary Materials.

21

27

Raw Ground Beef (80% lean) GENE-UP Salmonella with Alternative Confirmation

For the low inoculation level, 51 out of 132 test portions (POD CP of 0.39) were reported as presumptive positive by the candidate method with 50 out of 132 test portions (POD CC of 0.38) confirming positive. For the candidate method true positives, 49 out of 132 test portions (POD C of 0.37) were both presumptive positive and confirmed positive. For the reference method, 51 out of 132 test portions were reported as positive (POD R of 0.0.39). A dLPOD C value of -0.02 with 95% confidence interval of (-0.15, 0.12) was obtained between the candidate and reference method, indicating no statistically significant difference between the two methods. A dLPOD CP value of 0.01 with 95% confidence intervals of (-0.03, 0.04) was obtained between presumptive and confirmed results indicating no statistically significant difference. For the high inoculation level, 132 out of 132 test portions (POD CP of 1.00) were reported as presumptive positive by the candidate method with 130 out of 132 test portions (POD CC of 0.98) confirming positive. For the candidate method true positives, 130 out of 132 test portions (POD C of 0.98) were both presumptive positive and confirmed positive. For the reference method, 127 out of 132 test portions were reported as positive (POD R of 0.96). A dLPOD C value of 0.02 with 95% confidence interval of (-0.03, 0.09) was obtained between the candidate and reference method, indicating no statistically significant difference between the two methods. A dLPOD CP value of 0.02 with 95% confidence intervals of (-0.01, 0.04) was obtained between presumptive and confirmed results indicating no statistically significant difference. For the non-inoculated controls, 0 out of 132 samples (POD CP of 0.00) produced a presumptive positive result by the candidate method with 0 out of 132 test portions (POD CC and POD C of 0.00) confirming positive. For the reference method, 0 out of 132 portions were reported as positive (POD R of 0.00). A dLPOD C value of 0.00 with 95% confidence interval of (-0.03, 0.03) was obtained between the candidate and reference method, indicating no

22

28

statistically significant difference between the two methods. A dLPOD CP value of 0.00 with 95% confidence intervals of (-0.02, 0.02) was obtained between presumptive and confirmed results indicating no statistically significant difference. Detailed results of the POD statistical analysis for the GENE-UP Salmonella 2 method with alternative confirmation are presented in Table 2020.2A and Figures 1A – 1D. GENE-UP Salmonella with Traditional Confirmation For the low inoculation level, 51 out of 132 test portions (POD CP of 0.39) were reported as presumptive positive by the candidate method with 51 out of 132 test portions (POD CC of 0.39) confirming positive. For the candidate method true positives, 49 out of 132 test portions (POD C of 0.37) were both presumptive positive and confirmed positive. For the reference method, 51 out of 132 test portions were reported as positive (POD R of 0.0.39). A dLPOD C value of -0.02 with 95% confidence interval of (-0.15, 0.12) was obtained between the candidate and reference method, indicating no statistically significant difference between the two methods. A dLPOD CP value of 0.00 with 95% confidence intervals of (-0.04, 0.04) was obtained between presumptive and confirmed results indicating no statistically significant difference. For the high inoculation level, 132 out of 132 test portions (POD CP of 1.00) were reported as presumptive positive by the candidate method with 130 out of 132 test portions (POD CC of 0.98) confirming positive. For the candidate method true positives, 130 out of 132 test portions (POD C of 0.98) were both presumptive positive and confirmed positive. For the reference method, 127 out of 132 test portions were reported as positive (POD R of 0.96). A dLPOD C value of 0.02 with 95% confidence interval of (-0.03, 0.09) was obtained between the candidate and reference method, indicating no statistically significant difference between the two methods. A dLPOD CP value of 0.02 with 95% confidence intervals of (-0.01, 0.04) was obtained between presumptive and confirmed results indicating no statistically significant difference.

23

29

For the non-inoculated controls, 0 out of 132 samples (POD CP of 0.00) produced a presumptive positive result by the candidate method with 0 out of 132 test portions (POD CC and POD C of 0.00) confirming positive. For the reference method, 0 out of 132 portions were reported as positive (POD R of 0.00). A dLPOD C value of 0.00 with 95% confidence interval of (-0.03, 0.03) was obtained between the candidate and reference method, indicating no statistically significant difference between the two methods. A dLPOD CP value of 0.00 with 95% confidence intervals of (-0.02, 0.02) was obtained between presumptive and confirmed results indicating no statistically significant difference. Detailed results of the POD statistical analysis for the GENE-UP Salmonella method with traditional confirmation are presented in Table 2020.2B and Figures 2A – 2D. During the collaborative study, the candidate method was able to accurately detect and differentiate the target microorganism from high levels of competing background microflora from within the matrix. Several laboratories indicated that the high background microflora made identifying target organism difficult, even after sub-culturing in a secondary enrichment. These collaborators indicated the ease of use of the GENE-UP Salmonella method in detecting the pathogen would be very beneficial to their laboratory for matrices with high background microflora. Fifteen collaborators were solicited to participate in the study. One collaborator was unable to complete testing and did not submit results. A second collaborator did not perform the traditional confirmation procedure and therefore their data was not included. Two additional laboratories (1 and 10) were removed from data analysis due to contamination in their non- inoculated control samples. Discussion

24

30

When combining all 11 data sets used in the statistical analysis, 4 false positive results, 1 false negative result with the alternative confirmation procedure and 2 false negative results with the traditional confirmation procedure were obtained. For these discrepant results, 2 false positive results were obtained in the low inoculum level and 2 false positive results were obtained in the high inoculum level. All false negative results were obtained in the low inoculum level. The FP and FN rates were calculated as follows:

False Positive Rate = [FP/(True Positives + FP)] x 100

False Negative Rate = [FN/(True Negatives + FN)] x 100

For the study, a false positive rate of 2.2%, along with a false negative rate of 0.9% for the alternative confirmation and a false negative rate of 0.5% for the traditional confirmation were obtained. For the candidate method, variations observed within laboratory analysis (repeatability) and between laboratories (reproducibility) were the same, indicating consistency in the method’s performance by individual users and between users at different facilities. No statistically significant differences were observed between the presumptive and confirmed results (using either the alternative or traditional confirmation procedure) for the candidate method nor between the candidate and reference methods.

Recommendations

It is recommended that the GENE-UP ® Salmonella (SLM) method be adopted as Official First Action status for the detection of Salmonella species in a broad range of foods and select

25

31

environmental surfaces: 25 g test portions of fresh raw ground chicken, fresh raw chicken breast, fresh raw ground beef (80% lean), fresh raw fish, creamy peanut butter, vanilla ice cream, dry pet food and milk chocolate; for 30 mL test portions of chicken carcass rinse; for 100 g test portions of pasteurized liquid eggs and powdered egg; for 375 g test portions of fresh raw ground beef (80% lean), raw beef trim, raw ground pork, raw ground bison, whey protein powder, dry pet food, creamy peanut butter, garlic powder, bulk bagged Romaine lettuce, dark chocolate and milk chocolate; for stainless steel environmental sponges.

Acknowledgements

We would like to extend a sincere thank you to the following collaborators for their dedicated participation in this study: Jerri-Lynn Pickett, Courtney Watson & Liberty Madewell – Tyson/WBA Analytical; Springdale, AR Leslie Thompson – SGS Vanguard Sciences; North Sioux City, SD Janet Smith – Fieldale Farms; Baldwin, GA Kevin Miller – Kwik Trip ; La Crosse, WI Yuliana Ortiz, Diana Hernandez, Mike Erickson, Andrea Cipriani – Mérieux NutriSciences; Salinas, CA Srecko Prodanovic – Amway ; Ada, MI Hua Wang– FDA CFSAN; College Park, MD Sonal Patil, Ben Howard – Certified Laboratories; Aurora, IL Ryan Zimmerman, Thomas Donohue & LeAnne Hahn – Deibel Laboratories ; Madison, WI Dave Metzger & Thomas Hirsch – Accelerated Analytical Labs; Milwaukee, WI

26

32

David Simon and Reddy Bommineni – Florida Department of Agriculture and Consumer Service; Kissimmee, FL Dana Waggoner & Megan Davis – South Carolina Department of Health & Environmental Control; Columbia, SC Stephen Paranal & Karilyn Gonzalez – Food Microbiology Laboratories, Inc.; Cypress, CA

We would also like to extend a special thanks to the scientific affairs team at bioMérieux who contributed to the validation study: Patricia Rule, Michelle Keener, Vikrant Durant, Deborah Briese and Samoa Asigau.

27

33

References

(1) United States Department of Agriculture Food Safety and Inspection Service (2013) Salmonella Questions and Answers https://www.fsis.usda.gov/wps/portal/fsis/topics/food-safety-education/get-answers/food- safety-fact-sheets/foodborne-illness-and-disease/salmonella-questions-and-answers/ [Accessed November 2019] (2) Center for Disease Control and Prevention (2018) FoodNet 2017 Preliminary Data https://www.cdc.gov/foodnet/reports/prelim-data-intro-2017.html [Accessed November 2019] (3) Foodsafety.gov (2019) Salmonella https://www.foodsafety.gov/blog/sites/default/files/Salmonella-bynumbers.jpg [Accessed November 2019] (4) Official Methods of Analysis (2019) 21 st Ed., AOAC INTERNATIONAL, Rockville, MD, Appendix J. http://www.eoma.aoac.org/app_j.pdf [Accessed November 2019] (5) AOAC Performance Tested Method SM Program (2019) GENE-UP ® Salmonella 2 (SLM 2). PTM No. 121802 https://www.aoac.org/aoac_prod_imis/AOAC_Docs/RI/19PTM/19C_121802_BMGUS2. pdf [Accessed November 2019] (6) United States Department of Agriculture Food Safety and Inspection Service (2019) Microbiology Laboratory Guidebook 4.10 Isolation and Identification of Salmonella from Meat, Poultry, Pasteurized Egg, and Siluriformes (Fish) Products and Carcass and Environmental Sponges https://www.fsis.usda.gov/wps/wcm/connect/700c05fe-06a2- 492a-a6e1-3357f7701f52/MLG-4.pdf?MOD=AJPERES [Accessed November 2019]

28