森林脑炎病毒(TBEV)实时定量TaqMan PCR检测方法的建立

目的建立快速检测TBEV的实时定量TaqMan PCR方法。方法根据GenBank发表的TBEV全基因组序列资料，在其C基因和NS5基因区段设计TBEV的特异探针和引物，在E基因区段设计普通PCR引物。以MDJ01株作为待检毒株，黄病毒属的另外7株病毒用来评价检测体系的特异性。测定病毒的TCID50值并制备病毒拷贝数标准品，分别用于制作病毒滴度和拷贝数标准曲线。与常规PCR方法进行了灵敏度比较，并建立了病毒感染小鼠的检测模型。结果该检测方法的灵敏度可达到100拷贝／反应或0．1TCID50，是常规PCR方法的十倍。作为对照的黄热病毒疫苗株17D、登革1～4型病毒、日本脑炎病毒、西尼罗病毒Chin-01株结果均为阴性，证明该体系具有良好的特异性。经过多批次、不同浓度样品的重复检测，批内和批间变异系数均小于5％，表明该体系具有较好的稳定性和重复性。结论建立了一种灵敏、特异、简便易行的TBEV的TaqMan实时定量PCR检测方法，为TBEV的预防控制和诊断提供了一种技术手段。

Establishment of real-time quantitative TaqMan PCR assay for the detection of tick-borne encephalitis virus

Objective To develop a real-time quantitative PCR(RQ-PCR) assay based on TaqMan technology for rapid detection and quantification of tick-born encephalitis virus (TBEV) RNA. Methods According to all the TBEV genome sequences in GenBank, RQ-PCR primers and probes were designed in the conservative regions of TBEV C gene and NS5 gene. In addition, primers for conventional PCR were designed using E gene as target. The detective system was established and validated by using TBEV MDJ01 strain. In order to examine the specificity of the system, other viruses of flavivirus were assayed with the RQ-PCR simultaneously. The TBEV standard curve was drawn respectively by measuring TCID_ 50 titre and copy number. The sensitivity of RQ-PCR and the conventional PCR assays were compared, and TBEV infected mice model was reproduced for evaluation. Results The sensitivity of RQ-PCR assay was 100copies/reaction or 1 TCID_ 50 , which was 10 fold higher than conventional PCR. The results were all negative when used to detect other flavivirus including the yellow fever virus, dengue virus type 1, 2, 3 and 4, Japanese encephalitis virus, West Nile virus. The coefficient of variability was less than 5% from inter- and intra-assay showing that both the repeatability and stability of the system were good. Conclusion A sensitive, specific and convenient RQ-PCR method has been established, which is valuable for early detection of TBEV.