High-resolution Melting Curve Analysis Coupled with Fluorescence Real-time PCR for Simultaneous Detection of Four Food-borne Pathogenic Bacteria in Chicken
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Abstract:
A high-resolution melting curve analysis (HRM) technique was established for simultaneous and rapid detection of Salmonella, Listeria monocytogene, Campylobacter Staphylococcus aureus and Campylobacter jejuni in chicken meat. In this study, the fimY, hly, nuc and orfC specific genes of the above four bacteria were used as target genes to establish amultiple HRM-real time PCR detection system that can simultaneously detect a variety of food-borne pathogens. The specificity, sensitivity and reproducibility of the reaction and the artificially infected samples were evaluated. The established HRM-real time PCR detection system produced specific melting curves for Salmonella, Listeria monocytogene, Staphylococcus aureus and Campylobacter jejuni, with the TM values being 82.2 ℃, 78.2 ℃, 75.5 ℃ and 73.5 ℃, respectively. The sensitivity of the multiple HRM-real time PCR reaction system was 102 copies/mL for single pathogenic bacterium solution and 102 copies/mL for multiple pathogenic bacteria solution. The intra-CV value in the repeatability test of the reaction system was good between 0.03% and 0.32%, and the inter-CV value was between 0.45% and 2.12. The detection limits of the four bacteria for the artificially infected samples all reached 5 cfu/25 g. The results showed that the multiple HRM real-time PCR system can effectively detect and distinguish the above four common food-borne pathogens in poultry meat. The method has high specificity and sensitivity and good response repeatability, thus, is expected to play a role in the detection of food-borne pathogens.
