AOAC RI ERP Final Action Recommendations eBook

FINAL (Version 4)

DuPont™ BAX ® System Real-Time PCR Assay for Salmonella : Collaborative Study

reference culture methods. A total of 15 laboratories participated in this study, with 14 laboratories 1 reporting data for each matrix. Each collaborator received instructions for performing the study and 2 required materials prior to the start of the study. If necessary, training on the BAX® System was 3 provided to laboratory personnel by a DuPont representative. 4 This collaborative study was conducted in accordance with the AOAC INTERNATIONAL Methods 5 Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces 6 [4]. Frankfurter samples were evaluated against the USDA-FSIS reference method [1] as a paired study, 7 as the test and reference method enrichment protocols are identical. Orange juice samples were 8 evaluated against the FDA-BAM reference method [2] as an unpaired study, as the BAX® System method 9 uses an enrichment in proprietary media. Estimates of repeatability, reproducibility, and probability of 10 detection (POD) were evaluated. 11 12 Sample product was obtained from a local retail outlet and screened by the organizing laboratory to 13 identify any naturally-contaminating Salmonella and determine a total aerobic plate count. For each 14 sample type five analytical size portions (25 g for orange juice and 325 g for hot dogs) were screened for 15 Salmonella using the appropriate reference method. Although naturally contaminated samples would 16 have been preferred, all samples tested returned negative results for Salmonella . Therefore, each 17 sample matrix was artificially inoculated with a different serovar of Salmonella for use in this study. 18 Portions of each sample type were inoculated at levels that on the day of initiation of analysis produced 19 a high spike level (POD ~1.0 or approximately 5 cfu/test portion) and a low spike level (POD 0.25–0.75 or 20 0.2–2.0 cfu/test portion). Additional matrix was left uninoculated to serve as negative controls. 21 To inoculate frankfurter samples, a pure colony of Salmonella Typhimurium was transferred from 22 Trypticase Soy agar with 5% sheep’s blood (SBA) into Brain Heart Infusion broth (BHI) and incubated at 23 35°C for 18-24 hours. The inoculum was heat stressed in a 55°C water bath for 10 minutes to obtain a 24 percent injury of approximately 70% (as determined by plating onto selective Xylose Lysine 25 Desoxycholate agar and non-selective Tryptic Soy agar). The inoculation process started by inoculating 26 four small portions of the bulk matrix (all equal sizes). After each of these portions had been inoculated 27 dropwise with an 18-22 hr culture of the target organism, they were homogenized by hand. After 28 homogenization, all four portions were combined one at a time into a single container, homogenizing 29 the bulk material after each portion was added. The bulk lot was separated into two sampling 30 containers and analysts simultaneously prepared test portions from each container for both the 31 candidate and reference method (four analysts total, with two analysts working from each bin, one of 32 the analysts weighed out the candidate method samples, the other the reference method 33 samples). After 40 samples had been removed (20 for each method, candidate and reference) at 34 random locations throughout the sample bin, the matrix was re-homogenized by combining the material 35 from both containers into one and mixing thoroughly for the purposes of maintaining an even 36 distribution of the organism. 37 Test Sample Inoculation and Distribution

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