肠道病毒, EV71 及CoxA16 多重PCR 检测方法的研究.doc

肠道病毒、EV71及CoxA16多重PCR检测方法的研究 倪楠1*，候佩强2，张荣强3，陆娟娟4，韩彬5，孙涛6 （山东泰安市疾病预防控制中心，泰安 271018） 【摘要】 目的 建立多重聚合酶链反应（multiplex polymerase chain reaction）检测方法，同时快速从手足口病人粪便、咽拭子标本中检测肠道病毒（Pan-enterovirus，PE71型（Enterovirs 71 EV71）和柯萨奇病毒A组16型（Coxsachie virus,CoxA16）。方法设计三对寡核苷酸引物分别在同一体系中扩增肠道病毒（Pan-enterovirus，PE）、肠道病毒71型（Enterovirs 71 EV71）及柯萨奇病毒A组16型（Coxsachie virus,CoxA16）特异性基因片段，优化反应体系，测定该方法的灵敏性，并用乙型脑炎病毒、轮状病毒、单纯疱疹病毒Ⅰ、Ⅱ型检测其特异性。EV71（﹢）标本可见264bp和440bp两条特异性条带，CoxA16（﹢）标本可见440bp、550bp两条特异性条带，PE（﹢）EV71（﹣）CoxA16（﹣）标本可见440bp一条特异性条带。cDNA最低检出浓度分别为4.78ug/ml、2.36 ug/ml、43.27 ug/ml。乙型脑炎病毒、轮状病毒、单纯疱疹病毒Ⅰ、Ⅱ型提取核酸进行同体系、同条件多重PCR扩增未见条带该方法能在一个反应条件下同时检测PE、EV71、CoxA16，特异性强，灵敏度高，能用于临床诊断和流行病学调查。 【关键词】肠道病毒；EV71；CA16；多重PCR Study on a multiplex polymerase chain reaction assay for human enterovirus，human enterovirus 71 and coxsackievirus A16 NI NanHOU Pei-qiang2，ZHANG Rong-qiang3, LU Juan-juan4,HAN Bin5,SUN Tao6(Taian Center for Diseases Control and Prevention, Taian 271000, China) 【Absrtact】 Objective To establish a rapid and accurate method for the detection of PE, EV71 and CoxA16 from patients with hand foot and mouth disease(HFMD) by multiplex polymerase chain reaction. Methods Three pairs of Oligonucleotide primers were designed according to the specific sexogene of PE, EV71 and CoxA16 to amplify genetic fragments by multiplex-PCR. The condition of multiplex-PCR was optimized in which sensitivity was determined; specificity was evaluated using Japanese encephalitis virus, rotavirus and herpes simplex virus types 1 and 2. Results After multiplex RCR amplification, two specific bands with the size of 264bp and 440bp were detected in EV71 positive specimen. CoxA16 positive specimen witnessed two bands of 440bp and 550bp. In those specimens who showed PE positive, EV71 and CoxA16 negative, only one band of 440bp was detected. The lowest detectable concentration of virus cDNA was 4.78?g/ml, 2.36 ?g/ml, and 43.27 ?g/ml individually. With the same PCR amplification system and condition, no bands were detecte