肠道病毒、乙脑病毒和腮腺炎病毒多重RT-PCR检测方法的建立

Design and Construction of Multiplex RT-PCR Diagnostic Methods for Enterovirus,Japanese Encephalitis Virus and Mumps Virus

为建立针对肠道病毒(enterovirus,EV)、乙脑病毒(Japanese encephalitis virus,JEV)和腮腺炎病毒(mumps virus,MUV)的多重RT-PCR检测方法,分别选择肠道病毒的5′UTR基因、乙脑病毒的E基因和腮腺炎病毒M基因设计3对引物,建立同时检测肠道病毒、乙脑病毒和腮腺炎病毒的多重RT-PCR方法.以中国地区流行的与脑炎相关的麻疹病毒和风疹病毒cDNA为模板验证该检测方法的特异性,同时以梯度稀释的不同滴度的脊髓灰质炎病毒、乙脑病毒、腮腺炎病毒评估该检测方法的检出限.所建立的3种病毒的多重RT-PCR方法可同时或分别特异扩增肠道病毒、乙脑病毒和腮腺炎病毒的152 bp、429 bp和274 bp基因片段,基因序列分别与脊髓灰质炎病毒Sabin 1型5′UTR基因片段、乙型脑炎病毒SA-14-14-2株E基因片段及腮腺炎病毒S79基因片段序列一致;检出限分别达到78.1 CCID50/mL、312.5 PFU/mL、156.2 CCID50/mL;而对麻疹病毒和风疹病毒的扩增均为阴性.所建立的多重RT-PCR特异性和检出限良好,可用于上述3种脑炎病毒的快速检测.

In order to develop a rapid, sensitive and specific multiplex RT-PCR to simultaneously detect enterovirus （EV）, Japanese encephalitis virus （JEV） and mumps virus （MUV）, three sets of specific primers were selected and designed based on the alignment results of 5＇UTR gene of EV, E gene of JEV and M gene of MUV, and used in one PCR reaction. The PCR conditions were optimized. The specificity of the method was verified by using Measles virus and Rubella virus, which are encephalitis viruses prevalent in China, and the sensitivity of the method was verified by using poliovirus, JEV and MUV. Three specific PCR products, 152 bp for EV, 429 bp for JEV and 274 bp for MUV, were amplified, and their sequences were in accordance with EV, JEV and MUV respectively. The sensitivity of EV, JEV and MUV was 78.1 CCID50/mL, 312.5 PFU/mL and 156.2 CCIDsdmL respectively. None of Measles virus and Rubella virus was specifically amplified by multiplex RT-PCR. The multiplex RT-PCR could be valuable for rapid detection of EV, JEV and MUV.