逆转录-聚合酶链反应检测鼠肝炎病毒方法的建立

Establishment of the Detection Method of Mouse Hepatits Virus Using Reverse Transcriptase Polymerase Chain Reaction

建立特异、灵敏的逆转录聚合酶链反应（RT－PCR）技术，结合碱性磷酸酶标记的探针杂交检测鼠肝炎病毒（MHV），采用MHV－3，MHV－A59病毒株感染DBT细胞，37℃培养，待细胞出现病变时收集提取病毒RNA。依据MHV基因序列设计一对高度保守区特异性引物，进行RT－PCR扩增，结果可见147bp的鼠肝炎病毒产物特异扩增带。敏感性实验检测到10pg的鼠肝炎病毒RNA，同时用ELISA方法对照。结果提示应用RT—PCR技术结合探针杂交检测鼠肝炎病毒。具有简便、快速、灵敏等优势。本研究在国内尚未见报道。

A specific and sensitive method of Mouse Hepatits Virus (MHV) detection was establishedusing RT-PCR followed by hybridization with alkaline-phosphatase (AP) labelled probe. DBTcells infected with MHV-3 and MHV-A59 virus were incubated at 37℃, and the virus RNAswere collected after the cells began to get pathology. RT-PCR was perfomed with the primerdesigned according to the conservative sequences of the MHV gene, as a result, a 147hp specificamplification band was detected. As little as 10pg MHV RNA could be detected successfully usingthis method, but the titer was 1:64 using ELISA, which suggested that detection of MHV usingRT-PCR followed by hybridization with AP labelled probe was superior for its convenient, rapidand sensitive. This is the first time to report in our country.