4. AOACRIMicroMethods-2018Awards

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870  W allace et al . : J ournal of AOAC I nternational V ol . 97, N o . 3, 2014

determining the difference in POD values between the candidate and reference methods for each matrix and concentration (dLPOD C = POD C – POD R ). If the confidence interval of a dLPOD does not contain zero, then the difference is statistically significant at the 5% level. AOAC Official Method 2013.02 Salmonella species in a Variety of Foods and Environmental Surfaces BAX ® System Real-Time PCR Assay for Salmonella First Action 2013 [Applicable to the detection of Salmonella in a variety of foods, including raw ground beef (25 and 375 g), ground beef with soy (25 and 325 g), beef trim (25 and 325 g), frankfurters (325 g), shrimp (25 g), ground turkey (25 g), chicken wings (25 g), poultry rinse (30 mL), whole powdered (dried) eggs (25 g), shell eggs (1000 mL), fresh bagged lettuce (25 g), frozen peas (25 g), orange juice (pasteurized; 25 mL), cream cheese (25 g), nonfat dry milk (25 g), ice cream (25 g), peanut butter (25 g), cocoa (25 g), white pepper (25 g), milk-based infant formula (25 mL), and dry pet food (375 g), and on stainless steel, ceramic tile, and plastic surfaces.] See Table 2013.02 for a summary of results of the collaborative study. See Appendix 4, Tables 1–6 for detailed results of the collaborative study. Caution: Kits. —The reagents used in the BAX System

2925 mL BPW was added, and samples were incubated at 35°C for 18–24 h. For the test method, samples were tested directly from the BPW enrichment using the BAX System method. For the USDA-FSIS MLG reference method, 0.5 mL aliquots of each portion were transferred to 10 mL tetrathionate (TT) Hajna broth, and 0.1 mL sample was added to 10 mL modified Rappaport-Vassiliadis (mRV) broth. All secondary enrichments were incubated at 42 ± 0.5°C for 22–24 h (or in a water bath for 18–24 h). Secondary enrichments were streaked to brilliant green sulfa and either double modified lysine iron agar (LIA) or xylose lysine Tergitol TM 4 agar plates and incubated 35 ± 2°C for 18–24 h. Isolated colonies were transferred to triple sugar iron (TSI) agar and LIA slants and incubated 35 ± 2°C for 22–26 h. Salmonella colonies were confirmed using serological (Somatic O and poly H agglutination) and biochemical procedures according to USDA-FSIS MLG. For testing orange juice, each collaborator received 12 low-spike, 12 high-spike, and 12 uncontaminated 25 mL test portions blind-coded so that the contamination level was unknown to the collaborator. For the test method, samples were swirled with 225 mL BAX System MP media and incubated at 39–42°C for 22–26 h, then secondary enrichment was performed by transferring 10 µL primary enrichment to 500 µL prewarmed (37°C) BHI broth. Secondary enrichments were incubated at 37°C for 3 h, then tested with the BAX System method. For the FDA-BAM reference method, portions were swirled with 225 mL Universal Preenrichment Broth (UPB) and incubated at 35°C for 22–26 h. After primary enrichment, 1 mL of each enriched portion was transferred to 10 mL TT broth and 0.1 mL was transferred to 10 mL RV broth. RV tubes were incubated at 42 ± 0.2°C for 22–26 h using a circulating, thermostatically controlled water bath. TT tubes were incubated at 35 ± 2°C for 22–26 h. Secondary enrichments were streaked to bismuth sulfite, XLD, and Hektoen enteric agar plates and incubated at 35°C for 22–26 h. Isolated colonies were transferred to TSI and LIA slants and incubated 35 ± 2°C for 22–26 h. Salmonella colonies were confirmed using serological and biochemical procedures according to FDA-BAM. Data analysis was performed using each of the metrics below according to the format described in the AOAC INTERNATIONAL Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces and using the Least Cost Formulations, Ltd, AOAC Binary Data Interlaboratory Study Workbook (6). For the purposes of this evaluation, POD was defined as the number of positive outcomes divided by the total number of trials. POD was estimated with a 95% confidence interval for each of the following levels: candidate presumptive results (POD CP ); candidate confirmatory results (POD CC ); candidate method results based on the presumptive and confirmatory results (POD C ); and reference method results (POD R ). Candidate presumptive and confirmatory results were compared by determining the difference between the POD (dLOPD) values for each matrix and concentration (dLPOD CP = POD CP – POD CC ). If the confidence interval of a dLPOD does not contain zero, then the difference is statistically significant at the 5% level. Candidate and reference method results were compared by Statistical Analysis

should pose no hazards when used as directed. Dispose of lysate, PCR mixture, and other waste according to your site practices. laboratory personnel should operate the cycler/detector. Do not attempt to repair the instrument. Live power may still be available inside the unit even when a fuse has blown or been removed. Refer to the BAX System User Guide for maintenance procedures when cleaning the unit or changing a fuse. The heating block can become hot enough during normal operation to cause burns or cause liquids to boil. Wear safety glasses or other eye protection at all times during operation.  Enrichment broths. —All enrichment broths may contain varying pathogens whether they contain Salmonella or not and thus should be sterilized and disposed of using proper procedures following any culture-based confirmatory steps.  Reference cultures .—When handling reference Salmonella cultures, always follow appropriate biosafety containment procedures as provided by your standard laboratory site practices, Centers for Disease Control and Prevention (CDC), or Canadian Pathogen Safety Data Sheets and Risk Assessment.  Cycler/detector. —Only qualified

A. Principle The DuPont™ BAX System uses the polymerase chain reaction (PCR) to amplify a specific fragment of bacterial

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