肠道病毒、EV71及CoxA16多重PCR检测方法的研究  

Study on a multiplex polymerase chain reaction assay for human enterovirus, human enterovirus 71 and coxsackievirus A16

作　　者：倪楠[1] 侯佩强[1] 张荣强[1] 陆娟娟[1] 韩彬[1] 孙涛[1] 

机构地区：[1]山东省泰安市疾病预防控制中心,山东泰安271018

出　　处：《中国病原生物学杂志》2013年第5期439-441,共3页Journal of Pathogen Biology

基　　金：泰安市科学技术发展计划项目(No.20103020)

摘　　要：目的建立多重聚合酶链反应(multiplex polymerase chain reaction)方法,检测手足口病患者粪便、咽拭子标本中的肠道病毒(Pan-enterovirus,PE)、肠道病毒71型(Enterovirs71EV71)和柯萨奇病毒A组16型(Coxsachie virus,CoxA16)。方法设计3对寡核苷酸引物,分别在同一体系中扩增PE、EV71及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。结果对临床标本作多重PCR检测,EV71(+)标本可见264bp和440bp两条特异性条带,CoxA16(+)标本可见440p、550bp两条特异性条带,PE(+)EV71(-)CoxA16(-)标本可见440bp1条特异性条带。病毒cDNA最低检出浓度分别为4.78、2.36和43.27μg/ml。提取乙型脑炎病毒、轮状病毒、单纯疱疹病毒Ⅰ、Ⅱ型核酸,进行同体系、同条件多重PCR扩增,均为阴性。结论多重PCR方法能在一个反应体系和反应条件下同时检测PE、EV71、CoxA16,且特异性强,灵敏度高,可用于手足口病的临床诊断和流行病学调查。Objective To establish a rapid and accurate method for detection of pan-enterovirus （PE）, human enterovirus 71 （EV71）, and coxsackievirus A16 （CoxA16） from patients with hand, foot, and mouth disease （HFMD） using a multiplex polymerase chain reaction （PCR）. Methods Three pairs of oligonucleotide primers were designed to amplify gene fragments specific to PE, EV71, and CoxA16 using multiplex-PCR. Conditions for multiplex-PCR were optimized and its sensitivity was determined; its 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 a size of 264 bp and 440 bp were detected in EV71-positive specimens. CoxA16-positive specimens displayed two bands of 440 bp and 550 bp. In those specimens that were positive for PE and negative for EV71 and CoxA16, only one band of 440 bp was detected. The lowest detectable concentration of viral cDNA was 4.78 g/ml, 2.36 g/ml, and 43.27 g/ml respectively. The same PCR amplification system and conditions resulted in no bands from the amplified products of encephalitis B virus, rotavirus, and herpes simplex virus 1 and 2. Conclusion Multiplex-PCR is a simple, sensitive, and specific way to detect PE, EV71, and CoxA16 that could be used for clinical diagnosis and epidemiological investigation.

关 键 词：肠道病毒 EV71 CA16 多重PCR 

分 类 号：R383.24[医药卫生—医学寄生虫学]