5种脑炎人兽共患病病毒多重RT-PCR检测方法的建立

Establishment of multiplex PCR for the detection of five encephalitis zoonosis viruses

为建立同时检测流行性乙型脑炎病毒(JEV)、森林脑炎病毒(TBEV)、东方马脑炎病毒(EEEV)、西方马脑炎病毒(WEEV)和基孔肯雅病毒(CHIKV)5种人兽共患脑炎病病毒的多重RT-PCR方法,本研究根据GenBank登录的相关病毒基因序列设计特异引物,通过优化引物组合及PCR反应条件,建立可同时检测5种病毒的方法,扩增片段长度分别为411 bp(JEV)、945 bp(TBEV)、193 bp(EEEV)、545 bp(WEEV)和769 bp(CHIKV);该方法具有良好的特异性,对病毒核酸最低检测拷贝数分别为7.1×103、3.6×103、2.2×103、5.6×103和5.1×103。该方法具有特异性强、灵敏度高、操作简便等优点,为以上5种人兽共患脑炎病病毒提供快速检测手段。

The serious encephalitis symptoms both in human and in animals could be induced by one of the five encephalitic zoonosis viruses,including Japanese B encephalitis virus（JEV）,tick-borne encephalitis virus（TBEV）,eastern equine encephalitis virus（EEEV）,western equine encephalitis virus（WEEV） and Chikungunya virus（CHIKV）.A multiplex PCR method was developed for detecting the 5 virus with a sets of specific primers designed according to the published viral genome sequences.The PCR products for each virus was 411 bp（JEV）,945 bp（TBEV）,193 bp（EEEV）,545 bp（WEEV） and 769 bp（CHIKV）,with the detection limit of 7.1×103,3.6×103,2.2×103,5.6×103 and 5.1×103 of cDNA copies,respectively.This multiplex PCR method was proved to be a sensitive,specific method for rapid detection of the 5 zoonosis viruses.