5种猪源消化道传播病原体荧光定量PCR检测方法的建立

Establishment of Fluorescent Quantitative PCR Detection Method for Five Pathogens Transmitted by Digestive Tract in Swine

猪源人兽共患病不仅威胁公共卫生安全,还威胁生猪养殖业的健康发展;其中消化道传播是其主要的传播途径。为及时鉴定出经消化道传播的病原体,本试验建立了猪源大肠杆菌、沙门菌、单增李斯特菌和志贺菌的四重荧光定量PCR和戊型肝炎病毒荧光定量PCR检测方法,并优化反应体系和条件,实现5种病原体的同时检测;并利用荧光定量PCR与普通PCR对117份猪临床样品(病变组织、粪便和肌肉等)进行对比检测。结果显示,建立的荧光定量PCR能够在1.5 h内完成对大肠杆菌、沙门菌、单增李斯特菌、志贺菌和戊型肝炎病毒5种病原体的特异性检测,与其他常见细菌和病毒无交叉反应,检测极限值可达5个拷贝,标准曲线相关系数均不低于0.997,线性范围涵盖1×10^(1)~1×10^(9),批内和批间变异系数(CV)均低于3.16%。建立的荧光定量PCR与普通PCR方法检测结果的符合率达到95.73%~100%,具有较好的一致性。117份临床样品中大肠杆菌、沙门菌、单增李斯特菌、志贺菌和戊型肝炎病毒各自的阳性率分别为31.62%、17.95%、6.84%、5.13%和11.11%。结果表明,本试验所建立的荧光定量PCR方法灵敏、特异、稳定,能够同时、快速区分检测上述5种猪源消化道传播病原体,可为猪肉制品从产地到餐桌全环节样品的监测提供有力的技术支持。

Swine-origin zoonosis not only pose a threat to public health but also jeopardize the healthy development of the swine farming industry,with gastrointestinal transmission being a major route of dissemination.In order to promptly identify pathogens transmitted through the digestive tract in swine,this study established a quadruple fluorescent quantitative PCR method for the detection of swine-origin Escherichia coli(E.coli),Salmonella,Listeria monocytogenes,Shigella,and a fluorescent quantitative PCR method for detection of hepatitis E virus.The reaction system and conditions were optimized to achieve the simultaneous detection of these five pathogens.A comparative analysis was conducted using fluorescent quantitative PCR and conventional PCR on 117 clinical samples from pigs(including lesion tissues,feces,and muscles).The results showed that the established fluorescent quantitative PCR could achieve specific detection of E.coli,Salmonella,Listeria monocytogenes,Shigella,and hepatitis E virus within 1.5 hours.There were no cross-reactions with other common bacteria and viruses.The detection limit reached 5 copies,and the correlation coefficients of the standard curves were not less than 0.997.The linear range covered 1×10^(1)to 1×10^(9),and the intra-batch and inter-batch coefficients of variation(CV)were both less than 3.16%.The consistency rate between the results of the established fluorescent quantitative PCR and conventional PCR methods was 95.73%to 100%,demonstrating good agreement.Among the 117 clinical samples,the positive rates for E.coli,Salmonella,Listeria monocytogenes,Shigella,and hepatitis E virus were 31.62%,17.95%,6.84%,5.13%,and 11.11%,respectively.These results indicate that the established fluorescent quantitative PCR method is sensitive,specific,and stable,enabling simultaneous and rapid differentiation of the mentioned five swine-origin gastrointestinal pathogens.It provides robust technical support for the comprehensive monitoring of samples from the origin to the dining table in pork products.