Microbiology Methods for ERP Review 3-2020

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GENE ‐ UP ® SLM 2 Collaborative Study

July 2019

INTRODUCTION The goal of this collaborative study is to estimate the following performance parameters of the bioMérieux GENE ‐ UP ® Salmonella 2 (SLM 2): probability of detection (POD) and the difference in the POD at 3 contamination levels for both the candidate and reference methods. Test portions from 3 different contamination levels, including non—inoculated controls, will be sent to collaborators where they will perform the GENE ‐ UP SLM 2 method along with the corresponding reference method. Raw ground beef (80% lean) will be tested with 375 g test portion sizes. Description of the GENE ‐ UP Salmonella 2 Method The GENE ‐ UP Salmonella 2 (SLM 2) kit is designed for use with the GENE ‐ UP® Thermocycler. The GENE ‐ UP® SLM 2 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 nm, while the fluorescent signal for an internal amplification control is recorded in channel 705 nm. The software automatically interprets the results for the internal amplification 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. 1.1 The GENE ‐ UP SLM 2 method was validated according to AOAC Guidelines in a precollaborative study. The aim of this study was to demonstrate that the candidate method could detect Salmonella in a broad range of foods and select environmental surfaces as claimed by the manufacturer. During the precollaborative studies, several matrix studies were conducted. The candidate method was compared to the USDA – Food Safety Inspection Service (FSIS) Microbiology Laboratory Guidebook (MLG) Salmonella Chapter and the US Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Chapter 5 Salmonella reference methods using the current versions of the methods available at the time of validation. The candidate method was validated for the detection of Salmonella in 25 g test portions of fresh raw ground chicken, fresh raw chicken breast, 1.2 Summary of Precollaborative Study

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