TaqMan多重实时定量PCR快速检测3种常见食源性病原菌

Rapid Detection of Three Common Foodborne Pathogens by Multiplex Real-time Quantitative PCR Based on TaqMan Probes

建立一种可同时快速检测大肠杆菌O157:H7、单增李斯特菌和蜡样芽孢杆菌的多重实时定量PCR(qPCR)方法。依据大肠杆菌O157:H7 tir基因、单增李斯特菌mpl基因和蜡样芽孢杆菌entFM基因的保守序列分别设计特异性引物和TaqMan探针,建立多重qPCR反应体系,进行灵敏度、特异性和稳定性试验,同步检测人工染菌牛奶样品中的病原菌并与国家标准方法作对比。结果表明,建立的多重qPCR方法灵敏度高,最低检出限为12 CFU/mL;特异性强,只对3种目标菌进行PCR扩增;稳定性好,各重复性试验中Ct值的变异系数<1%;所有受污染样品阳性检出率均为100%,与国家标准方法检测结果一致,且检测周期缩短至6 h。本研究建立的TaqMan多重qPCR方法能同时快速、准确地检测乳品中的大肠杆菌O157:H7、单增李斯特菌和蜡样芽孢杆菌,为食品安全提供技术支撑。

The study was to establish a multiplex real-time quantitative PCR(qPCR)method based on TaqMan probes for simultaneous detection of Escherichia coli O157:H7,Listeria monocytogenes and Bacillus cereus.Specific primers and TaqMan probes were designed according to the conservative sequences of tir,mpl and entFM genes,corresponding E.coli O157:H7,L.monocytogenes and B.cereus,respectively,and a multiplex qPCR assay was presented to test their sensitivity,specificity and stability.The assay was used to detect pathogenic bacteria in contaminated milk samples,comparing with the national standard method.The experimental results showed that the multiplex qPCR assay exhibited high sensitivity,strong specificity and good stability,corresponding the minimum detection limited being 12 CFU/mL,only three kinds of target bacteria being amplified in the multiple-bacteria sample and the variation coefficient of repetitive amplification being less than 1%,respectively.The positive detection rate of bacteria-contaminated samples was up to 100%through the multiplex qPCR assay,consisting with the national standard method,and the testing can be fast finished within 6 h.It confirmed that the multiplex qPCR assay can rapidly and accurately detect the three common foodborne pathogens,namely E.coli O157:H7,L.monocytogenes and B.cereus in contaminated milk,and provided effective technical supports for food security.