【摘要】 目的:通过调查近年来我国肠道病毒EV-71型和柯萨奇病毒A16型流行株的全基因组序列,建立一种能够获得我国肠道病毒序列的通用扩增方法,为今后的手足口病流行病学分析、致病机理研究等打下基础。方法:收集我国近5年各地报道的肠道病毒流行株全基因组序列作为参考序列进行比对分析,在保守区设计通用引物,利用3’RACE、长距离PCR扩增及简并引物扩增肠道病毒全基因组序列,采用Ion Torrent PGM二代测序仪对扩增产物进行深度测序,以对扩增方法进行验证和评价。结果:通过比对肠道病毒流行株序列设计了通用扩增引物,经二代测序实验获得了肠道病毒全基因组序列;以系列比例模拟混合病毒感染,该扩增方法能够同时获得2株肠道病毒的全基因组序列;能够完整地揭示肠道病毒重组情况。结论:建立了针对我国近年来肠道病毒流行株的通用全基因组扩增方法,在病毒培养液中肠道病毒的提取与扩增中显示了较高的灵敏度,能够反映混合病毒感染、重组病毒的情况。更多还原

【Abstract】 Objective: To collect the genome-wide sequences of enterovirus EV-71 and coxsackievirus A16 strains in recent years in China, and establish a universal amplification method for obtaining Chinese enterovirus sequences, laying the foundation for the epidemiological analysis and pathogenesis of hand, foot and mouth disease in the future. Methods: The whole genome sequences of enterovirus epidemic strains reported in China in the past five years were collected as reference sequences to be compared and analyzed, and then, the universal primers were designed in the conserved regions. The enterovirus genome-wide sequence was amplified using 3’RACE,long-distance PCR amplification, and degenerate primers. The amplified products were subjected to deep sequencing using an Ion Torrent PGM second generation sequencer to verify and evaluate the amplification method. Results: Universal amplification primers were designed by comparing the sequences of enterovirus epidemic strains,and the whole genome sequence of enterovirus was obtained by second-generation sequencing. The amplification method was capable of simultaneously obtaining the whole genome sequences of two enteroviruses from a mixed virus infection by a series of ratios, therefore, it can reveal the combination of enterovirus. Conclusion: A universal genome-wide amplification method for enterovirus epidemic strains in China has been established. It shows high sensitivity in the extraction and amplification of enteroviruses in virus culture fluids, and can reflect mixed virus infection and combined virus.更多还原