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

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.