Validation of the Multiplex PCR Assays for Detection of Salmonella spp. and Cronobacter sakazakii on Stainless Steel and Sealed Concrete Surfaces Compared with FDA-BAM Reference Methods

BACKGROUND: Rapid laboratory detection methods need to be tested against reference methods to confirm their efficiency and suitability. OBJECTIVE: In an unpaired study, the IEH Salmonella-Cronobacter spp. and Cronobacter sakazakii-Salmonella Multiplex PCR Assays' performance was compared with the FDA-BAM reference methods Chapter 5 for Salmonella and Chapter 29 for Cronobacter sakazakii detection on stainless-steel (SS) and sealed concrete (SC) surfaces. METHODS: Seeking a shorter enrichment time for the candidate method, the Salmonella-Cronobacter broth enrichments for 18, 20, 22, and 24 h at 35 °C were analyzed with the IEH Multiplex PCR Assays. Two pathogen cocktails were prepared: one including S. Typhimurium ATCC 19585, S. Senftenberg 775W, and S. Enteritidis PT30, and a second one including C. sakazakii MEI 27583, C. sakazakii ATCC 29544, and C. sakazakii ATCC 29004. The Salmonella and C. sakazakii cocktails were independently inoculated onto separate SS and SC following the AOAC guidelines. Additionally, a competing microorganism was added at a concentration 10-100-times higher than that of the pathogens. RESULTS: The candidate method yielded zero false negatives and zero false positives, with 100% sensitivity and 100% specificity for both SS and SC surfaces, regardless of the inoculation level (high, low, and uninoculated) and the enrichment time (i.e. 18, 20, 22, and 24 h for Salmonella, and 20, 22, and 24 h for C. sakazakii). The candidate method successfully detected both target microorganisms on both surface types with a minimum enrichment of 20 h. Statistical analysis using the probability of detection (POD) confirmed the equivalency between the candidate method and the reference methods at either inoculation level or enrichment time (P > 0.05). CONCLUSION: The IEH Salmonella-Cronobacter spp. and Cronobacter sakazakii-Salmonella Multiplex PCR Assays demonstrated performance comparable to the FDA-BAM reference methods for the detection of Salmonella and C. sakazakii on SS and SC, with a significantly shorter turnaround time.