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目的研究北京市丰台区手足口病(Hand-Foot-Mouth Disease,HFMD)患者肠道病毒A组71型(Enterovirus Group A Type 71,EVA71)分离株的基因特征。方法使用荧光定量逆转录-聚合酶链反应(Real-time Reverse Transcript-Polymerase Chain Reaction,r RT-PCR)方法对北京市丰台区2013年3~9月采集于HFMD患者的咽拭子标本进行EVA71核酸检测,对检测结果为EVA71阳性的标本,用人横纹肌肉瘤细胞进行病毒分离,对分离到的EVA71毒株进行VP1编码区基因扩增,并对扩增产物进行核苷酸序列测定和分析。结果在81份咽拭子标本中,有26份经r RT-PCR方法检测为EVA71核酸阳性,分离到6株EVA71,VP1编码区有6个散在位点(43、58、164、184、240、249)存在共变异,氨基酸组合模式为赖氨酸-丙氨酸(Alanine,Ala)-天冬氨酸-丝氨酸-苏氨酸-缬氨酸,在170位氨基酸均为Ala,在145位氨基酸均为谷氨酸。核苷酸序列分析结果表明,6株EVA71的VP1编码区核苷酸同源性为94.3%~100.0%,与C4a亚型代表株的VP1编码区核苷酸同源性最高(90.0%~97.6%)。进化树图分析结果显示,北京市丰台区EVA71分离株与C4亚型代表株处于同一分支,并在C4a进化分支的不同簇中。结论北京市丰台区EVA71分离株为C4亚型C4a进化分支。
Objective To investigate the genetic characteristics of Enterovirus Group A Type 71 (EVA71) isolated from patients with Hand-Foot-Mouth Disease (HFMD) in Fengtai District of Beijing. Methods The throat swab specimens of HFMD collected in Fengtai District of Beijing from March to September 2013 were analyzed by Real-time Reverse Transcript-Polymerase Chain Reaction (r RT-PCR) Nucleic acid test, the test results were positive for EVA71 specimens, human rhabdomyosarcoma cells were used for virus isolation, the isolated strain of VP71 VP1 gene amplification, and amplification products were sequenced and analyzed. Results Among 81 pharyngeal swab specimens, 26 were positive for EVA71 by RT-PCR and 6 were isolated, and 6 were scattered in the VP1 coding region (43, 58, 164, 184, 240 , 249). The amino acid pattern was Alanine, Ala-Asp-Ser-Thr-Val, the amino acids at position 170 were all Ala, The amino acids are glutamic acid. The results of nucleotide sequence analysis showed that the homology of the VP1 coding region of the six strains of EVA71 was 94.3% -100.0%, which was the highest (90.0% -97.6) with the VP1 coding region of C4a subtype %). The results of phylogenetic tree analysis showed that the isolate of strain EVA71 in Fengtai District of Beijing was in the same branch with the representative strain of C4 subtype and was in different clusters of C4a evolutionary branch. Conclusion Feng71 district in Beijing is the C4 subtype C4a evolutionary branch.