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目的了解新疆肠道病毒71型(EV71)分离株VP1区基因特征。方法选取2011-2013年新疆部分地州手足口病病例标本,临床标本或其病毒培养物经实时荧光PCR鉴定后,通过RT-PCR法进行VP1区编码基因扩增,并对扩增产物进行序列测定,所得序列用ChromasPro 1.7.4,BioEdit7.1.11和MEGA5.1软件进行序列校正、拼接、整理和分析,并与EV71各型及亚型参考序列构建基于VP1序列的系统进化树。结果新疆EV71分离株之间核苷酸和氨基酸序列同源性在92.8%~100.0%和97.9%~100.0%,22株病毒株与安徽阜阳和山东临沂C4a基因参考株最为相近,核苷酸和氨基酸序列序列同源性在93.9%~98.6%和98.6%~99.6%。而与A、B基因型参考株差异较大,核苷酸和氨基酸序列序列同源性在82.1%~84.8%和95.9%~98.3%。直接用临床标本进行核酸提取、扩增和序列测定的阳性率为30.0%,低于用病毒培养物的阳性率(100.0%)。结论 2011-2013年新疆EV71分离株均为C4a基因型,与安徽和山东分离的EV71毒株可能有相同的起源。直接用临床标本进行核酸提取、扩增和序列测定可用于肠道病毒的分型鉴定。
Objective To understand the characteristics of VP1 gene of enterovirus 71 (EV71) isolates in Xinjiang. Methods From 2011 to 2013, some cases of HFMD in Xinjiang were identified. Real-time PCR was used to identify the clinical samples or virus cultures. RT-PCR was used to amplify the gene encoding VP1 region. The amplified products were sequenced The phylogenetic tree based on the VP1 sequence was constructed with the sequences of ChromasPro 1.7.4, BioEdit 7.1.1 and MEGA5.1. Results The nucleotide and amino acid sequences of EV71 isolates in Xinjiang were 92.8% -100.0% and 97.9% -100.0%, respectively. Most of the 22 isolates were the same as the reference strains of C4a in Fuyang, Anhui Province and Linyi in Shandong Province. The nucleotide and The homologies of amino acid sequences ranged from 93.9% to 98.6% and from 98.6% to 99.6%. However, there were significant differences between A and B genotype reference strains. The nucleotide and amino acid sequence homologies were between 82.1% -84.8% and 95.9% -98.3%. The positive rate of direct nucleic acid extraction, amplification and sequencing with clinical samples was 30.0%, lower than the positive rate of virus culture (100.0%). Conclusion The EV71 isolates in Xinjiang from 2011 to 2013 are all C4a genotypes, and EV71 isolates from Anhui and Shandong may have the same origin. Direct clinical samples for nucleic acid extraction, amplification and sequencing can be used for typing of enterovirus identification.