黄病毒属TaqMan探针荧光定量RT-PCR检测方法建立

Establishment of TaqMan real-time PT-PCR for detection of flavivirus

目的建立用于检测黄病毒属的TaqMan探针荧光定量RT-PCR方法。方法从GenBank中检索黄病毒属代表株的全基因序列,通过DNAstar进行序列比对和blast进行保守序列搜索和分析,用Beacon Designer 7.0软件进行引物和TaqMan探针设计,以乙脑毒株MB090509 RNA为模板,优化反应条件并应用于现场蚊虫标本检测。结果确定黄病毒属TaqMan探针荧光定量RT-PCR反应条件为:45℃15 min,95℃10 min,95℃10 s→54℃45 s,40次循环,引物和探针终浓度为200 nmol/L。灵敏度为常规RT-PCR方法 100倍,检出限2.9×10-3噬斑形成单位(PFU)/mL,与甲病毒属、东南亚十二节段RNA病毒属、布尼亚病毒属均无交叉反应。从161份现场蚊虫标本中检测出11份阳性标本,经病毒分离、测序均被证实为乙脑病毒。结论本研究建立的黄病毒属TaqMan探针荧光定量RT-PCR方法具有广泛应用价值。

Objective To establish a method for screening flavivrus by TaqMan real-time retro-trancriptase（RT）-PCR.Methods The complete genome sequences of yellow fever virus（YFV）,Japanese encephalitis virus（JEV）,dengue virus（DENV）,and West Nile virus（WNV） were searched from GenBank.The conserved sequences of the viruses were aligned and blasted with DNAstar software.Then several pairs of primers and probes were designed with the Beacon Designer 7.0.The RNA of JEV strain MB090509 was used as positive control to establish the best detection condition for TaqMan real-time RT-PCR.And then 161 pools of mosquitoes were detected by the TaqMan real-time RT-PCR method.Results The best detection conditions of real-time RT-PCR for flavivirus were the primers and detection probe concentration of 200 nmol/L and 40 amplifying cycles of 45 ℃ for 15 min,95℃ for 10min,95℃ for 10 sec,and 54℃ for 45 sec.The TaqMan real-time RT-PCR method showed high sensitivity and specificity.The sensitivity of the method was 100 times higher than that of RT-PCR.The low detection limit of the mehtod was 2.9×10-3PFU /mL.There was no cross-reaction with Getah virus,Banna virus,and Bunyamwera virus.Eleven pools were detected being positive for JEV among 161 pools of mosquitoes with the the traditional virus isolation and identification.Conclusion The TaqMan real-time RT-PCR method established could be generally used for flavivirus detection.