西安地区手足口病患儿咽拭子中EV71的检测及分离鉴定

Detection and identification of EV71 in throat swab specimens of children with hand-foot-mouth disease in Xi'an

目的对2009年儿科门诊22例疑似手足口病患儿的咽拭子标本进行EV71病毒检测及分离鉴定。方法采集的咽拭子标本分别接种于人横纹肌瘤细胞(RD细胞),盲传3代,若细胞圆缩、分散、胞浆内颗粒增加,即出现致细胞病变效应(CPE),运用RT-PCR方法检测标本和细胞培养物上清液中的EV71,提取RNA,分离鉴定。结果标本直接用RT-PCR法扩增,阳性率为31.8%(7/22),经细胞培养后出现CPE的阳性率为45.5%(10/22),培养后经RT-PCR再检测阳性率为68.2%(15/22),所分离的病毒株基因序列同GENEBANK中报道的EV71(序列号EU812461)序列完全一致。结论病毒分离和RT-PCR 2种检测方法联合应用可以提高肠道病毒EV71的阳性检出率,为临床诊断提供有力依据。

Objective To detect and identify the EV71 virus in throat swabs specimens of children suspected with hand-foot-mouth disease.Methods The throat swabs specimens taken from children with hand-foot-mouth disease were inoculated with RD cells,blindly transferred to the third generation for observing the cytopathic effect（CPE）,that is cells becoming round,scattered and cytoplasm particles increased.RT-PCR was used to detect the EV71 virus from specimens and cell culture supernatant,and the total RNA was extracted for identification later.Results The positive rate of throat swabs specimens was 31.8%（7/22） and increased to 68.1%（15/22） in the inoculated specimens.45.4%（10/22） of the inoculated specimens showed CPE.The nucleotide sequence of the isolated virus was the same to the EU812461 in the NCBI GENBANK.Conclusions The application of viral isolation plus RT-PCR can enhance detection rates of EV71,which can offer a powerful way to the clinical diagnosis.