3种鼠源致病菌多重PCR检测方法的建立

Establishment and application of multiplex PCR procedure for detection of three pathogenic bacteria from mice

本研究以沙门菌组氨酸转运操纵子hut基因、肺炎克雷伯菌外膜蛋白Pho E基因以及金黄色葡萄球菌耐热核酸酶nuc基因作为靶基因,建立了同时检测这3种致病菌的多重PCR方法,并对混有3种致病菌的盲肠内容物分别采用传统细菌分离培养法和PCR方法进行检测,同时对混有3种致病菌的粪便样品进行PCR检测。该方法能扩增出目的片段分别为495 bp、333 bp、279 bp。在优化条件下,多重PCR同时检测沙门菌、肺炎克雷伯菌及金黄色葡萄球菌的敏感度分别为1.23 pg、1.9 pg、1.185 ng DNA。分离培养法对沙门菌、肺炎克雷伯菌及金黄色葡萄球菌的最低检出限分别为1×10~2CFU、1×10~0CFU和1×10~2CFU。混合有3种致病菌的盲肠内容物和粪便,经增菌培养后,PCR法的最低检出量均为1×10~0CFU。本研究建立的多重PCR检测方法具有特异性及灵敏度高、操作简单、周期短的优点,适用于实验小鼠多种致病菌的快速检测和监控。

The purpose of this paper is to establish a multiplex PCR method for simultaneous detection of Salmonella spp.,Klebsiella pneumonia and Staphylococcus aureus.A multiplex PCR assay targeting hut gene,Pho E gene,andnuc gene,which encode the histidine transport operon of Salmonella spp.,the outer membrane phosphoporin of Klebsiella pneumonia and the thermostable nuclease of Staphylococcus aureus,respectively,was developed for simultaneous detection of the three pathogenic bacteria.The traditional bacterial culture method and PCR assay were used to detect the cecum content which was contaminated with the three kinds of pathogenic bacteria,meanwhile,while feces contaminated with three kinds of pathogenic bacteria were detected by PCR assay.The PCR assay could amplify the target bands of 495 bp,333 bp and 279 bp.Under optimized conditions,the sensitivity of the multiplex PCR for Salmonella spp.,Klebsiella pneumonia,Staphylococcusaureus was 1.23 pg,1.9 pg and 1.185 ng DNA,respectively.The minimum detection limit of culture was 1×10^2 CFU for Salmonella spp.,1×10^0 CFU for Klebsiella pneumonia and 1×10^2 CFU for Staphylococcus aureus,while the minimum detection limit of PCR assay after enrichment culture for the cecum content and feces mixed with three kinds of pathogenic bacteria was 1×10^0 CFU.A multiplex PCR method that has the advantages of high sensitivity and specificity,operability and rapidness has been established in this study.The PCR method can be used for rapid diagnosis and monitoring of multiple pathogenic bacteria in laboratory mice.