Isolation, Identification And Establishment Of Real-time PCR Assay For Japanese Encephalitis Virus Genotype Ⅰ

Objective:Japanese encephalitis is a serious influence on human health disease. Although the JE cases has declined, but regional is still growing, we can assess the prevalence of JE with surveillance in the region. Mosquitoes are the major JEV vector. Under the surveillance of mosquitoes arboviruses virus and Japanese encephalitis, they can take appropriate precautions.Methods:15 mosquito samples from Yunnan Jinghong inoculated BHK-21 and C6/36 cell monolayers, cell appear to be cytopathic then extraction of its viral RNA. cDNA was performed to PCR identifition. The positive DNA were cloned into pMD18-T, sending the Shanghai sequenced. Simultaneously, PCR was identify of Getah virus, Sindbis, and Chikungunya virus,then sequenced too. On this basis, a standard Type I JE gene plasmids was constructed, quantitative PCR method based on SYBR Green I dye was established.Results:JE gene sequenced was type I, then applied to quantitative PCR standard plasmids. The establishment of a standard curve showed good repeatability, the melting curve of a single peak, no non-specific amplification occurs. Compared with the traditional method, RT-PCR SYBR Green I real-time PCR has 10 times higher sensitivity and good specificity.Conclusion:JE gene detected type I, and constructed rapid detection JE type I detection method. Preliminary experiments have verified its excellent sensitivity and reproducibility, laid the foundation for future epidemiological JE investigation.