Development of TaqMan Probe-based Real-Time PCR Assays for Rapid Detection of Clostridium perfringens, Staphylococcus aureus, and Salmonella spp. in Meat and Evaluation of Effect of Brief Enrichment on Their Sensitivity
摘要
Foodborne diseases caused by bacterial pathogens are of major public health and zoonotic concern. The objective of the present study was to develop real-time TaqMan PCR assays for the detection and quantification of important bacterial foodborne pathogens, including Clostridium perfringens, Staphylococcus aureus, and Salmonella spp. For this purpose, primers and probes were designed from conserved regions of the target genes (cpa for C. perfringens, nuc for S. aureus, and invA for Salmonella spp.) and TaqMan assays were standardized. The analytical sensitivity of the developed real-time TaqMan assays using gel-purified PCR amplicons was determined to be 2.8 copies/μL, 3.5 copies/μL, and 7.0 copies/μL of DNA for C. perfringens, Salmonella spp., and S. aureus, respectively. The analytical sensitivity of the TaqMan assays was 10- to 1000-fold higher than that of conventional endpoint PCR. The standard curves showed good linearity with R2 = 0.99 for all the pathogen-specific TaqMan assays developed and the assays were found to be reliable and reproducible. In spiking studies, the limit of detection (LoD) of the developed TaqMan assays under un-enriched conditions was 1.2 × 105 CFU/g, 3.2 × 108 CFU/g and 3.3 × 104 CFU/g of meat for C. perfringens, Salmonella spp., and S. aureus respectively. After 6 h of enrichment, the LoD considerably improved to 1.2 CFU/g, 320 CFU/g, and 3.3 CFU/g of meat, respectively. The study highlights the need and importance of the enrichment step in the detection of FBPs. The developed real-time TaqMan assays may serve as rapid laboratory tools for the detection and quantification of C. perfringens, S. aureus, and Salmonella spp. in meat.