建立高分辨熔解曲线技术同时快速检测鸡肉中沙门氏菌、单增李斯特菌、金黄色葡萄球菌和空肠弯曲菌。本研究分别以上述4种菌的fimY、hly、nuc和orfC特异性基因为靶基因，建立能同时检测多种食源性致病菌的多重HRM-real time PCR检测体系，并对反应的特异性、灵敏度、重复性以及在人工染菌样品进行了评价。所建立的HRM-real time PCR检测体系对沙门氏菌、单增李斯特菌、金黄色葡萄球菌和空肠弯曲菌均产生特异性熔解曲线，Tm值分别为82.2 ℃、78.2 ℃、75.5 ℃和73.5 ℃；多重HRM-real time PCR反应体系检测单一致病菌菌液的灵敏度均为102拷贝数/mL，检测多种致病菌菌液的灵敏度为102拷贝数/mL；该反应体系重复性的试验内CV值在0.03%~0.32%之间，试验间CV值在0.45%~2.12%；人工染菌样本对4种菌的检测限均能达到5 CFU/25 g。结果表明该多重HRM-real time PCR检测体系可对上述4种禽肉中常见致病菌进行有效的检测与区分，特异性强，灵敏度高，反应重复性好，有望在食源性致病菌检测分析中发挥作用。

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.

国家自然科学青年基金项目(31700005)；扬州市社会发展项目（YZ2020060）；江苏省属公益类科研院所自主科研经费(BM2018026)；江苏省现代农业产业技术体系建设专项资金资助(JATS[2020]359)