TaqMan PCR检测乙型脑炎病毒方法的建立及应用

Establishment and application of a TaqMan PCR 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,探讨其用于临床诊断的可行性。方法:根据GenBank发表的JEV全基因组序列资料分析结果,在其3′端非编码区(UTR)设计JEV特异的引物与探针,验证检测体系的特异性、灵敏性和稳定性,并用于临床乙脑病人样本的检测。结果:引物、探针具有良好的特异性,用该TaqMan PCR法检测JEV减毒株SA14-14-2灵敏度可达1 TCID50/ml。稳定性分析表明,同一样本重复检测5次,Ct值变异系数均小于5%。在此基础上,实时荧光PCR检测了10份临床上确诊为乙脑患者的标本(特异性IgM阳性),6份阳性,阳性率为60%。在7份特异性IgM阴性、临床上疑似乙脑的患者中,2份TaqMan PCR结果为阳性,阳性率为28.6%。此方法从RNA提取到检测结果仅需1.5 h。结论:本研究建立了一种灵敏、特异、简便易行的JEV TaqMan PCR检测方法,为乙型脑炎的鉴别诊断提供了一种技术手段。

Objective：To explore a molecular diagnostic method for Japanese encephalitis virus（JEV） based on TaqMan PCR and to evaluate the possibility of TaqMan PCR in clinical diagnosis of Japanese encephalitis（JE）.Methods：JEV genomic sequences were analyzed and JEV specific primers and probe in 3′-UTR were co-designed by Primer Express 2.0.Specificity,sensitivity and stability analysis tests were performed by JEV strains and other flavivirus or encephalitis associated virus.The constructed TaqMan PCR method was primarily used to clinical samples test for JEV.Results：Test showed that the method appeared highly specific and sensitive.The lowest detecting limit was 1 TCID50/ml.Stability test showed that co-efficient variables were all less than 5% in 5 different samples.In further experiments with clinical specimens,10 samples of IgM（＋）JE patients,7 samples of suspected JE patients with IgM（-） were tested by TaqMan PCR.Specific amplification occurred in 6/10 and 2/7 of the patients.Primary application showed that TaqMan PCR for JEV was sensitive,specific,easy and fast.The test could be completed in 1.5 hours.Conclusion：One sensitive,specific and quick-performing method was established for JEV based on TaqMan PCR and it could be applied for laboratory diagnosis of JEV.