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目的从手足口病患儿粪便中分离肠道病毒71型(EV71),对其进行全基因组测序,了解秦皇岛市EV71全基因组基因特征,为手足口病的综合防治提供依据。方法从秦皇岛市各医院收集手足口病患儿粪便标本,进行EV71检测,阳性标本进行病毒分离及核酸提取,通过RT-PCR进行全基因组序列扩增,通过电泳、测序及序列拼接一系列操作分析其基因特征。结果用提取的核酸扩增出7 406bp的目的基因片段测序后与已记录的EV71相应序列进行比对完全一致,且测序峰图较好,可信度也较高;各基因片段间通过核苷酸序列拼接后得出,秦皇岛市医院EV71全基因组序列长7 406bp,编码区各基因片段的长度分别为:2A450bp、2B297bp、2C987bp、3A258bp、3B66bp、3C549bp、3D1 386bp、VPl 891bp、VP2 762bp、VP3 726bp、VP4 207bp。结论秦皇岛市EV71分离株为C4a型全基因组序列长7 406bp,可为当地手足口病的防控提供参考。
Objective To isolate EV71 from feces of hand-foot-mouth disease patients and perform genome-wide DNA sequencing to understand the genomic characteristics of EV71 gene in Qinhuangdao and provide a basis for comprehensive prevention and treatment of HFMD. Methods Stool samples from hand, foot and mouth disease patients were collected from hospitals in Qinhuangdao for EV71 detection. The positive samples were isolated from virus and nucleic acids were extracted. The whole genome sequence was amplified by RT-PCR and analyzed by electrophoresis, sequencing and sequence splicing Its genetic characteristics. Results The nucleotide sequence of 7 406 bp amplified by the extracted nucleic acid was completely consistent with that of the corresponding sequence of EV71 sequenced, and the peak sequence of sequencing was better and the reliability was higher. The nucleotide sequence of EV71 in Qinhuangdao Hospital was 7 406bp. The length of each gene fragment in the coding region was 2A450bp, 2B297bp, 2C987bp, 3A258bp, 3B66bp, 3C549bp, 3D1 386bp, VP1 891bp, VP2 762bp, VP3 726bp, VP4 207bp. Conclusion The complete genomic sequence of C4a genotype in EV71 isolate of Qinhuangdao City is 7 406 bp in length, which may provide a reference for the prevention and control of HFMD in China.