通用多重不对称PCR结合寡核苷酸芯片检测7种食源性致病菌  

An Oligonucleotide Microarray Coupled with Universal Primer-Multiplex Asymmetric PCR for Detection of Seven Foodborne Pathogens

作　　者：王小强[1] 袁军[1] 韩锐郡[1] 营思思 WANG Xiaoqiang;YUAN Jun;HAN Ruijun;YING Sisi(Xi’an Center for Disease Control and Prevention, Xi’an 710054, China)

机构地区：[1]西安市疾病预防控制中心,陕西西安710054

出　　处：《食品科学》2017年第8期290-295,共6页Food Science

基　　金：陕西省自然科学基础研究计划项目(2015JM3112);西安市社会发展引导计划--医学研究项目(SF1516(2))

摘　　要：应用通用多重不对称聚合酶链式反应(polymerase chain reaction,PCR)和寡核酸芯片技术建立一种同时检测7种常见食源性致病菌的方法。每种致病菌上游或下游引物5’端连接一段异源的共有序列。以荧光标记的该共有序列作为通用引物,与限制性特异引物经一步多重不对称PCR同时获得所有目标菌的单链标记靶序列,可被芯片上固定的特异性寡核苷酸探针捕获。通过芯片扫描、分析荧光信号完成检测。标准菌株检测结果证实,该方法可特异地检测单一和混合感染的目标菌,基因组DNA的检测灵敏度为0.1~1 pg。95份模拟污染和零售食品样本芯片检测结果与常规的分离与生化鉴定及荧光定量PCR结果一致。建立的寡核苷酸芯片方法可为快速、特异、灵敏及高通量地鉴定食源性致病菌提供一种有效的检测手段。A new method that combined universal primer-multiplex asymmetric PCR and oligonucleotide microarray technology was developed for the simultaneous detection of seven common foodborne pathogens including Listeria monocytogenes,Salmonella,E.coli O157:H7,Staphylococcus aureus,Yersinia enterocolitica,Vibrio parahaemolyticu and a standard strain of Escherichia coli.The5'-end of forward or reverse primer specific to each pathogen was linked with a non-homologous common sequence.Target fragments of all seven pathogens were enriched and labeled simultaneously by one-step multiplex asymmetric PCR using the Cy3-labeled common sequence as the universal primer.The labeled single-stranded amplicons were captured by the specific oligonucleotide probes immobilized on microarrays,followed by microarray scanning and analysis of fluorescence signal intensity.The results for reference strains indicated that the assay could unambiguously discriminate all seven pathogens in single and multiple infections,and it had a detection sensitivity of0.1-1pg of genomic DNA.Ninety-five artificially contaminated samples and retail food samples were tested by this assay,and the results agreed with those obtained with traditional culture methods and real-time PCR.This developed oligonucleotide microarray assay can provide a useful method for rapid,specific,sensitive and high-throughput detection of foodborne pathogens.

关 键 词：食源性致病菌 通用引物 多重不对称PCR 寡核苷酸芯片 

分 类 号：Q789[生物学—分子生物学] R155.5[医药卫生—营养与食品卫生学]