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目的:对2008年分离自河南新乡手足口病患者粪便标本的病原进行分离鉴定及全基因组序列测定,并同相关毒株序列进行同源性比对和进化分析,以了解新分离病毒基因组序列特征及可能的传播来源。方法:将手足口病患者粪便标本接种敏感细胞进行分离传代培养,制备抗原片进行间接免疫荧光检测,采用特异性引物进行PCR鉴定,将病毒基因组分为8个片段进行RT-PCR扩增,扩增产物纯化后进行PCR测序。通过末端重叠序列拼接成全长病毒基因组序列,利用生物信息学软件对序列进行比对分析,绘制进化树。结果:测序获得的3株EV71病毒基因组全长均为7 405 nt,VP1氨基酸序列同源性达100%。Blast分析表明F3(F8)-Henan-08毒株均与安徽阜阳2008年分离的EV71毒株同源性最高,而F4-Henan-08毒株与我国台湾省2004年分离的EV71毒株同源性最高。序列进化分析表明,新分离毒株属于C4基因亚型。结论:新分离的河南EV71毒株与安徽阜阳分离的毒株可能具有相同来源。
OBJECTIVE: To isolate and identify the pathogen of stool specimens from hand, foot and mouth disease patients in Xinxiang, Henan Province in 2008 and to determine the genome-wide sequence of the virus, and compare homology and evolutionary analysis with related strain sequences to understand the sequence characteristics of newly isolated virus genomes And possible sources of communication. Methods: Stool specimens of hand, foot and mouth disease were inoculated with sensitive cells for subculturing. Antigen fragments were prepared for indirect immunofluorescence detection. The specific primers were used for PCR identification. The viral genome was divided into 8 fragments for RT-PCR amplification. PCR products were purified by PCR. The full-length viral genome was spliced by the overlapping sequences of the end and the sequences were aligned by bioinformatics software to draw the phylogenetic tree. Results: The total length of the 3 strains of EV71 viruses sequenced was 7 405 nt, and the homology of VP1 amino acid sequence was 100%. Blast analysis showed that F3 (F8) -Henan-08 strains had the highest homology with the EV71 strain isolated in Fuyang, Anhui Province in 2008, while the F4-Henan-08 strain was homologous with the EV71 strain isolated in Taiwan Province of China in 2004 The highest sex. Sequence analysis showed that the newly isolated strains belonged to C4 subtype. Conclusion: The newly isolated strains of EV71 in Henan and Fuyang in Anhui may have the same source.