Validation of the Thermo Scientific™ SureTect™ Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus PCR Assay for the Detection of Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus in Seafood Matrixes: AOAC Performance Tested MethodsSM 022301.

BACKGROUND: The Thermo Scientific™ SureTect™ Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus PCR Assay method is a real-time PCR method for the multiplex detection of Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus in seafood. OBJECTIVE: The Thermo Scientific SureTect Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus Assay was evaluated for AOAC Performance Tested MethodsSM certification. METHOD: Inclusivity/exclusivity, matrix, product consistency/stability, and robustness studies were conducted to assess the method's performance. For the matrix study, the method was validated using the Applied Biosystems™ QuantStudio™ 5 Real-Time PCR Food Safety Instrument and the Applied Biosystems™ 7500 Fast Real-Time PCR Food Safety Instrument against the U.S. Food and Drug Administration Bacteriological Analytical Manual, Chapter 9 (2004), Vibrio and ISO 21872-1:2017 Microbiology of the food chain-Horizontal method for the determination of Vibrio spp.-Part 1: Detection of potentially enteropathogenic Vibrio parahaemolyticus, Vibrio cholerae, and Vibrio vulnificus reference methods. RESULTS: Matrix studies showed equivalent or superior performance of the candidate method compared to the reference method and, overall, no difference between presumptive and confirmed results, except for one matrix due to high background flora. The inclusivity/exclusivity study correctly identified/excluded all strains analyzed. Robustness testing showed no statistically significant differences in assay performance under varied test conditions. Product consistency and stability studies demonstrated no statistically significant differences between assay lots with different expiration dates. CONCLUSIONS: The data presented show that the assay constitutes a rapid and reliable workflow for the detection of V. cholerae, V. parahaemolyticus, and V. vulnificus in seafood matrixes. HIGHLIGHTS: The SureTect PCR Assay method allows for fast, reliable detection of stipulated strains in seafood matrixes with results obtained in as little as 80 min post-enrichment.

Validation of the Thermo Scientific SureTect™ Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect™ Escherichia coli STEC Identification PCR Assay for the Detection of Escherichia coli O157:H7 and the Escherichia coli STEC Serotypes (O26, O45, O103, O111, O121, O145) from Fresh Raw Spinach, Fresh Baby Leaves, Fresh Cut Tomatoes, Frozen Raw Beef, Raw Beef Trim, and Beef Carcass Sponges: AOAC Performance Tested MethodSM 012102.

BACKGROUND: The Thermo Scientific SureTect™ Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect Escherichia coli STEC Identification PCR Assay are real-time PCR kits for the rapid detection of E. coli O157:H7 and non-E. coli O157 Shiga toxin-producing E. coli (STEC) serotypes (O26, O45, O103, O111, O121, O145) from fresh raw spinach, fresh baby leaves, fresh cut tomatoes, frozen raw beef, raw beef trim, and beef carcass sponges. OBJECTIVE: Both assays were evaluated for AOAC®Performance Tested MethodsSM certification. METHODS: Detection and confirmation inclusivity/exclusivity, matrix, product consistency and stability, and robustness studies were conducted. In the matrix studies, the candidate method was validated against United States and international reference methods for STEC serotypes. RESULTS: Matrix studies showed no statistically significant differences between the candidate and reference method results when analyzed by probability of detection. For each inclusivity/exclusivity study, all inclusivity strains and no exclusivity strains were detected by either kit. Robustness testing demonstrated that the identification assay performed reliably despite method deviations; however, although not statistically significant, the screening assay performance was impacted. Product consistency and stability testing demonstrated no statistically significant differences between kit lots and storage time points. CONCLUSION: The data presented show that both assays constitute a rapid and reliable workflow for the detection and confirmation of E. coli O157:H7 and stipulated non-E. coli O157:H7 STEC serotypes from the tested matrixes. HIGHLIGHTS: Results are obtained in 80 min post-enrichment with both assays run simultaneously, allowing for the detection and confirmation of STEC within a single workflow.

Validation of the Thermo Scientific™ SureTect™Staphylococcus aureus PCR Assay for the Detection of Staphylococcus aureus in Dairy Matrixes: AOAC Performance Tested MethodSM 052101.

BACKGROUND: The Thermo Scientific™ SureTect™Staphylococcus aureus PCR Assay is a real-time PCR assay for the detection of Staphylococcus aureus in dairy samples. OBJECTIVE: The Thermo Scientific SureTect Staphylococcus aureus PCR Assay was evaluated for AOAC Performance Tested MethodSM certification. METHODS: Inclusivity/exclusivity, matrix studies, product consistency and stability, and robustness testing were conducted to assess the method's performance. For the matrix study, the method was validated on the Applied Biosystems™ QuantStudio™ 5 Real-Time PCR instrument and the Applied Biosystems 7500 Fast Real-Time PCR instrument against the ISO 6888-3:2003 Microbiology of food and animal feeding stuffs-Horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species)-Part 3: Detection and MPN technique for low numbers, and the U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Ch. 12, Staphylococcus aureus, 2016, reference methods. RESULTS: Matrix studies showed no statistically significant differences between the candidate and reference methods or between presumptive and confirmed results. The inclusivity/exclusivity study correctly identified/excluded all strains analyzed. Robustness testing showed no statistically significant difference in assay performance after set method parameter deviations, and product consistency and stability studies demonstrated no statistically significant differences in performance between kit lots at different expiration points. CONCLUSION: The data presented show that the assay is a rapid and reliable workflow for the detection of S. aureus from dairy matrixes. HIGHLIGHTS: The PCR assay allows for fast, reliable detection of S. aureus in dairy matrixes with results obtained in as little as 80 min post enrichment.