人类肠道病毒分子生物学分型鉴定方法的建立及应用

Identification and typing of human enteroviruses using an RT-PCR assay

目的建立人类肠道病毒(HEV)分子生物学分型鉴定方法,用于临床分离肠道病毒的分型鉴定。方法根据已知各型别人类肠道病毒基因的核苷酸序列和文献资料,分别合成HEV种属特异性和分型鉴定引物;提取病毒RNA,采用逆转录聚合酶链反应(RT-PCR)扩增病毒cDNA,通过琼脂糖凝胶电泳和核苷酸序列分析,鉴定病毒并分型,并与血清中和试验相互验证。结果采用种属特异性引物鉴定18株疑似HEV毒株,其中17株阳性;型特异性引物鉴定结果:18株病毒中4株为科萨奇病毒A24型,3株为科萨奇病毒B3型;科萨奇病毒B2、A9、A15型、埃可病毒3、6、7、9、11、14、33型和鼻病毒9型各1株,分型结果与中和试验相符。结论建立的人类肠道病毒分子生物学分型鉴定方法,具有特异性强、敏感性高、快速简便等优点,可以直接鉴定并分型诊断人类肠道病毒感染,适用于人类肠道病毒感染的实验室诊断和流行病学研究。

Objective To establish a molecular detection and typing assay for identification and typing of human enteroviruses （HEV） which is suitable for clinical detection and epidemiologic research. Methods Using both primers specific for HEV genus and HEV typing primers and reverse transcription polymerase chain reaction （RT-PCR） the authors detected preliminarily HEV by agarose gel electrophoresis and then identified serotype through nucleotide sequence analysis of RT-PCR amplicons.The monospecific antisera neutralization was applied to validate the typing results.Results The serotype of 18 suspicious HEV samples was identified： 4 Coxackievirus type A24（CVA24）, 3 CVB3, 1 CVB2, 1 CVA9, 1 CVA15, 1 Echovirus type 3（E3）, 1 E6, 1 E9, 1 E11,1 E14, 1 E33 and 1 Rhinovirus type 9. The result was validated by monospecitive antisera neutralization. Conclusion This RT-PCR assay for HEV detection and typing may be suitabe for clinical detection and epidemic research since this method is sensitive and specific for direct identification and typing of HEV.