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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 identify pathogenic types of enteroviruses associated with hand-foot-mouth disease by molecular typing based on VP1 region gene sequencing. Methods The VP1 region of enterovirus was sequenced by reverse transcription-polymerase chain reaction (RT-PCR), and the amplified products were sequenced. Chromas Pro 1.7.6, MEGA 5.05 And other bioinformatics software sequence alignment and splicing, the sequence submitted to Enterovirus Genotyping Tool (0.5) identified type. Results Forty other cases of hand, foot and mouth disease with other enterovirus (non-EV-A71 and non-CVA16) nucleic acid were identified by commercial real-time RT-PCR kit, 31 Coxsackie A CVA6) and two Coxsackievirus A (CVA10) strains. For the remaining seven samples to be identified, the VP1 gene sequences were amplified and sequenced by using enterovirus-specific primers and analyzed to identify Coxsackie virus 4 strains of group A (CVA4), 9 strains of group A (CVA9), 1 strain of E14 (E-14), and EV-A71. Conclusion The typing method of VP1 based on the sequence of VP1 gene has good sensitivity, simplicity and rapidness. It can be used in the laboratory diagnosis and related research of HFMD and other enterovirus infections.