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目的采用逆转录聚合酶链反应(RT-PCR)检测2008年山东省部分地区手足口病(HFMD)患者肠道病毒71型(EV71),对毒株进行诊断及基因特征初步分析。方法以肠道病毒特异引物对EV71进行RT-PCR扩增,经琼脂糖凝胶电泳鉴定阳性标本,纯化后与pGEM-T载体连接,转化大肠埃希菌DH5α,筛选后测序,所得序列与我国阜阳、深圳、台湾株及美国EV71标准流行株的核苷酸序列在线进行BLAST比对,分析其同源性。结果42份标本中检出EV71 33例(78.57%),所得序列经种系进化分析,与阜阳2008年HFMD暴发分离株同源性为98.7%~99.1%,与深圳1998年HFMD散发分离的EV71毒株同源性为93.4%~93.8%,与台湾1998年HFMD病例分离毒株同源性为91.2%~91.6%,与美国EV71毒株同源性为79.2%~79.7%。有9株EV71的563位苏氨酸(T)替代了EU703813的异亮氨酸(I)。结论EV71 C4亚型是2008年山东省部分地区HFMD暴发流行的病原,同源性分析表明山东省部分地区EV71分离株与阜阳、深圳及台湾地区分离株有较高的同源性,提示该病毒在中国大陆有较广泛的传播。
Objective To detect the enterovirus 71 (EV71) in hand, foot and mouth disease (HFMD) patients in some areas of Shandong province in 2008 by reverse transcription polymerase chain reaction (RT-PCR). Methods EV71 was amplified by RT-PCR with enterovirus-specific primers. The positive samples were identified by agarose gel electrophoresis and purified. The positive samples were ligated with pGEM-T vector and transformed into Escherichia coli DH5α. The sequences were screened and sequenced. Fuyang, Shenzhen, Taiwan and the United States EV71 standard strains of the nucleotide sequence of the BLAST online comparison, analysis of its homology. Results 33 cases (78.57%) of EV71 were detected in 42 samples. The phylogenetic analysis showed that the sequence of EV71 was 98.7% -99.1% with HFMD outbreak in Fuyang in 2008 and EV71 The homology of the strains was 93.4% -93.8%. The homology between them was 91.2% -91.6% with Taiwan isolates of HFMD in 1998 and 79.2% -79.7% with that of the USA. The threonine (T) at position 563 of 9 EV71 replaced the isoleucine (I) of EU703813. Conclusion EV71 C4 subtype is the etiological agent of HFMD outbreak in some areas of Shandong Province in 2008. The homology analysis indicated that the EV71 isolates in some areas of Shandong Province have higher homology with Fuyang, Shenzhen and Taiwan isolates, suggesting that the virus In mainland China have a wider spread.