基于VP1区基因序列测定的肠道病毒分型方法在手足口病病原鉴定中的应用

Application of EV classification and identification on basis of VP1 region nucleotide sequence in pathogenic identification of HFMD

目的应用基于VP1区基因序列测定的分子分型方法鉴定手足口病相关肠道病毒病原型别。方法使用肠道病毒VP1区种属特异性,采用逆转录—聚合酶链式反应(RT-PCR)方法扩增VP1区基因序列,对扩增产物进行序列测定,运用Chromas Pro1.7.6、MEGA 5.05等生物信息学软件进行序列核对及拼接,将序列提交肠道病毒分型网站(Enterovirus Genotyping Tool 0.5)鉴定型别。结果 40份其他肠道病毒(非EV-A71、非CVA16)核酸阳性的手足口病病例临床标本,先行采用商品化实时荧光RT-PCR试剂盒鉴定出31份柯萨奇病毒A组6型(CVA6)、2份柯萨奇病毒A组10型(CVA10);对其余待鉴定的7份样本,采用肠道病毒特异性引物进行VP1基因序列扩增和测序并分析,鉴定出柯萨奇病毒A组4型(CVA4)4株和A组9型(CVA9)、埃可病毒14(E-14)各1株,另1株为EV-A71。结论基于VP1区基因序列测定的肠道病毒分型方法具有较好的敏感性和简便、快速的特点,可用于手足口病及其他肠道病毒感染的实验室诊断和相关的研究。

Objective To conduct classification and identification of human enterovirus（EV） samples of hand, foot and mouth disease（HFMD） with molecular classification method basing on VP1 region nucleotide sequence. Methods According to primers specific for EV genus and EV serotypies and reverse transcription polymerase chain reaction（RT-PCR）, VP1（virus capsid protein gene region 1） genes were amplified and sequenced, and then sequences were performed by bioinformatics software Chromas Pro1.7.6 and MEGA 5.05, then sent to Enterovirus Genotyping Tool for classification and identification. Results Of all 40 other EV（non-EV-A71, non-Coxackievirus A16） samples, 31 of Coxackievirus A6（CVA6） and 2 of Coxackievirus A10（CVA10） were previously confirmed by using commercial real time RT-PCR kits. Among 7 suspicious samples of the residual, 4 of Coxackievirus A4（CVA4）, 1 of Coxackievirus A9（CVA9）, 1 of enteric cytopathogenic human orphan virus（E-14） and 1 of EV-A71 were identified and typed. Conclusions The method of EV classification and identification basing on VP1 region sequence is sensitive, simple and rapid. It could be used for laboratory diagnosis and related studies focus on HFMD and some other EV infectious diseases.