Establishment of RT-PCR and SYBR Green Real-time PCR for detection of Flavivirus in mosquito

Objective To establish a method for screening the Flavivrus spp.by SYBR Green Real-time PCR.Methods The universal primers for Flavivrus were used,and JEV cDNA and DEN cDNA were used as templates to develop and assess the screening methods including RT-PCR and SYBR Green Real-time PCR.The sensitivity of the two methods were also evaluated.Results The sensitivity of SYBR Green Real-time PCR was 100 times higher than those of RT-PCR.The minimum amount of virus RNA detected by SYBR Green Real-time PCR was 0.5×10-2 PFU/ml.Conclusion RT-PCR and SYBR Green Real-time PCR might be useful in detection of JEV and DEN.SYBR Green Real-time PCR was more sensitive than RT-PCR.As the primers were universal for Flavivirus,it can be used for screening and detecting of Flavivirus spp.