乙型脑炎病毒TaqMan PCR检测方法的建立及初步应用

Construction and primary application of a TaqMan PCR detection method for detection of Japanese encephalitis virus

目的利用TaqMan PCR技术,建立乙型脑炎病毒(Japanese encephalitis virus,JEV)实时荧光PCR检测方法,初步应用于蚊虫媒介的JEV监测。方法根据GenBank发表的JEV全基因组序列资料分析结果,在其NS5基因区段设计JEV特异的引物与探针;优化Mg2+浓度和引物浓度比例,并利用11株JEV和7株相关病毒株考核检测体系的保守性、特异性、灵敏性和稳定性;利用体外转录的病毒RNA和定量的病毒样品,建立相应的定量分析模型;初步应用于蚊虫媒介的监测与检测分析。结果Mg2+优化浓度为5mmolL,上下游引物浓度比例为4∶1,引物探针具有良好的保守性和特异性,灵敏度为10PFUml,稳定性分析表明,同一样品Ct值的重复检测5次,变异系数均小于5%;绘制两种标准曲线,构建了JEV基因拷贝数、病毒滴度为分析指标的定量分析模型,检测蚊虫标本结果显示,TaqMan PCR方法较病毒分离法敏感、快捷、简便。结论本研究建立了一种灵敏、特异、简便易行的JEVTaqMan PCR检测方法,为蚊虫媒介检测奠定了基础,为乙型脑炎的预防控制和脑炎相关疾病的诊断与鉴别诊断提供了一种技术手段。

Objective To construct a molecular diagnostic method for Japanese encephalitis virus（JEV） based on TaqMan PCR. Methods Twenty-two JEV genomic sequences were analyzed and JEV specific primers and probe in NS5 were co-designed by Primer Express 2.0. Mg^2＋ concentration and primer ratio were optimized. Specificity, sensitivity and stabihty analysis tests were performed by 11 different JEV strains（ genotype Ⅰ and Ⅲ ） and 7 other flavivirus or encephalitis associated virus; sensitive analysis was tested by titered virus stock. Two quantitative analysis models were constructed using viral RNA transcripts and titered virus samples. The constructed TaqMan PCR method was primarily used to mosquito samples test for JEV. Results Mg^2＋ was optimized at 5 mmol/L, ratio of forward and reverse primers at 4：1. Test showed that the primers and probe were highly conservative and specific. The low detecting hmit was 10 PFU/ml. Stability test showed that co-efficient variables were all less than 5% in 4 different samples. Two different standard curves were drawn, thus two quantitative analysis models were built for virus genomic copy and virus titers. Primary application showed that TaqMan PCR for JEV is more sensitive, easier and faster to perform the traditional virus isolation and identification. Conclusion One sensitive, specific and quick-performing method was established for JEV based on TaqMan PCA; it could applied for JE diagnosis and vector surveillance.