Simultaneous Detection of Salmonella, Listeria monocytogenes, and Staphylococcus aureus by Multiplex Real-Time PCR Assays Using High-Resolution Melting

A method combining multiplex real-time polymerase chain reaction (PCR) with high-resolution melting (HRM) analysis for rapid and specific simultaneous detection of Salmonella, Listeria monocytogenes, and Staphylococcus aureus was developed. The method included a melting-curve analysis of products and was evaluated by specificity, sensitivity and reproducibility analyses. Sensitivity and reproducibility analyses was both conducted by genomic DNA extracted from serial dilutions for each target pathogen. Assays with artificially inoculated and naturally contaminated samples after enrichment were also conducted. In the specificity test, there was no nonspecific amplification of the 44 nontarget pathogens, whereas the actual T-m values were 79.38 +/- 0.14, 82.54 +/- 0.15, and 77.36 +/- 0.14 degrees C for Salmonella, L. monocytogenes, and S. aureus, respectively. The sensitivity of the method was 3.5x10(2) CFU ml(-1) for Salmonella and L. monocytogenes and 3.5x10(3) CFU ml(-1) for S. aureus. The coefficients of variation of Tm values ranged 0.51-1.03 % for Salmonella, 1.63-2.11 % for L. monocytogenes, and 0.75-2.17% for S. aureus in intraassay, and ranged 0.81-2.43% for Salmonella, 1.97-2.35 % for L. monocytogenes, and 0.93-3.93 % for S. aureus in interassay. The detection limit in artificially inoculated samples (n=50) was 5 CFU (25 g)(-1) food for the three tested pathogens. In the naturally contaminated samples (n=120), Salmonella DNA was detected by HRM, sequencing, and conventional culture-based methods at a positive rate of 25.00, 25.00, and 24.17 %, respectively; the corresponding rates for L. monocytogenes were 14.17, 14.17, and 14.17 %, respectively, while those for S. aureus were 16.7, 16.7, and 16.7 %, respectively.