实时荧光PCR在食源性致病菌监测中的应用研究

Exploratory Development of Real-Time Fluorescence PCR Assay in Monitoring Foodborne Pathogens

目的建立沙门菌、志贺菌、单核细胞增生李斯特菌、大肠埃希菌O157:H7、金黄色葡萄球菌5种致病菌的实时荧光PCR检测方法,并在食源性致病菌监测工作中推广应用。方法将菌株及样品经培养基增菌后,用热裂解法提取DNA,使用荧光定量PCR反应试剂盒,对该检测方法进行特异性验证,并在2006-2007年间,同时应用实时荧光PCR和传统方法对890份各类实际工作监测标本进行比较分析。结果实时荧光PCR方法对19株不同种类标准菌株符合率为100%;对用传统方法检测分离到的5种食源性致病菌的符合率分别为:沙门菌96.61%,单核细胞增生李斯特菌92.30%,大肠埃希菌O157:H7、志贺菌、金黄色葡萄球菌均为100%;对890份监测标本检测结果表明,实时荧光PCR法对食品及临床标本中食源性致病菌的检出率略高于传统培养法,差异无统计学意义,而实时荧光PCR法可在3～36h内对目标样品作出结果判断。结论实时荧光PCR方法成功应用于食源性致病菌的检测,具有快速、特异和灵敏的特点,可作为食物中毒等突发公共卫生事件处置和重大活动食品安全保障工作的有效技术支撑。

Objective To develop real-time fluorescence PCR assay for detecting Salmonella,Shigella,Listeria monocytogenes,EHEC O157：H7,Staphylococcus Aureus in monitoring foodborne pathogens.Method DNAs of strains and sample were extracted on boiling lyses method after cultivation over night.The specificity of the real-time PCR assay was identified by using PCR kit to amplify DNA.The detection rate of the real-time PCR assay and the traditional culture method were compared for 890 food samples tested during 2006-2007.Results In the detection of 19 standard strains,the coincidence rate of real-time PCR assay was 100%.In the detection of five pathogens isolated from food monitoring by traditional method,the coincidence rate of real-time PCR assay was 96.61% in Salmonella,92.30% in Listeria monocytogenes,and 100% in Shigella,EHEC O157：H7,Staphylococcus aureus.The detection rate of real-time PCR assay for 890 food samples was slightly higher than traditional methods,though the difference was nonsignificant.The testing results could be obtained in 3-36 h by real-time PCR assay.Conclusion Real-time PCR assay is rapid,sensitive and specific for rapid diagnosis of pathogens in monitoring food poisoning.It could provide new feasible technical support for the surveillance and safeguard food safety at public health emergencies and major assembly events.