AOAC RI ERP Final Action Recommendations eBook

Collaborative Study Approved Protocol Expert Review Panel Use Only

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1.0 2 3 The purpose of this collaborative study is to determine the performance of the candidate method 4 among collaborators for the detection of Salmonella in a variety of foods. Estimates of repeatability, 5 reproducibility, probability of detection (POD), and Relative Limit of Detection (RLOD) will be evaluated. 6 Collaborators will analyze 2 matrices at 3 contamination levels comparing the performance of the 7 candidate method to appropriate reference culture methods. The BAX® System Real-Time PCR Assay for 8 Salmonella is the candidate method. 9 10 1.1 BAX® System Real-Time PCR Assay for Salmonella Introduction The BAX® System Real-Time PCR Assay for Salmonella uses the Polymerase Chain Reaction (PCR) to amplify a specific fragment of bacterial DNA, which is stable and unaffected by growth environment. The fragment is a genetic sequence that is unique to the genus Salmonella , thus providing a highly reliable indicator that the organism is present. The BAX® System simplifies the PCR process by combining the requisite primers, polymerase and nucleotides into a stable, dry, manufactured tablet already packaged inside the PCR tubes. After amplification, these tubes remain sealed for the detection phase, thus significantly reducing the potential for This automated BAX® System method uses fluorescent detection to analyze PCR product. One PCR primer for each target (one Salmonella- specific target and an internal control) contains a fluorescent dye (two different dyes, one for each target) as a constituent of the primer as well as a quencher (the uni-molecular combination of a primer, fluorescent dye and quencher constitute a Scorpion™ Probe). When incorporated into a PCR product, the dye and quencher are spatially separated, which causes an increase in emission signal. The BAX® System measures the magnitude and characteristics of fluorescent signal change. An analysis by the BAX® System software algorithm then evaluates that data to determine a positive or negative result which is contamination with one or more molecules of amplified PCR product.

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Summary of the Performance Tested Method /Pre-collaborative study

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The BAX® System Real-Time PCR Assay for Salmonella was certified by the AOAC Research Institute in August, 2012 and designated Performance Tested Method SM (PTM) #081201. No significant differences were reported for detection of Salmonella in the matrices tested when comparing the BAX method results to the following standard reference culture procedures; U.S. Department of Agriculture-Food Safety and Inspection Service Microbiological Laboratory Guidebook (USDA-FSIS MLG) Chapter 4.05 (1), U.S. Food and Drug Administration Bacteriological Analytical Manual (FDA BAM) Chapter 5 (2), and Health Canada Compendium of Analytical Methods (HC CAM) MFHPB-20 (3). The matrices validated in the PTM study, along with the appropriate reference method used for comparison, include the following: raw ground beef (25

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