日本乙型脑炎病毒GⅢ型标准品质粒的构建

Construction of Japanese Encephalitis Virus GⅢStandard Plasmid

目的构建检测日本乙型脑炎病毒(JEV)GⅢ型拷贝数的质粒标准品,利用该质粒标准品定量检测病毒的拷贝数。方法通过设计引物经反转录聚合酶链反应扩增JEV相对保守区序列(prM结构蛋白),得到的目的片段克隆连接到pMD19-T载体上,从而获得JEV质粒标准品。随后,通过实时荧光定量聚合酶链反应(RT-qPCR)得到质粒标准品的标准曲线方程以及对JEV患者血清中病毒拷贝数进行检测。结果构建的JEV质粒标准品的RT-qPCR结果显示:相同浓度复孔间Ct值差异小(<0.5),熔解曲线峰值单一。得到的标准曲线方程为y=-0.2889 x+11.516,R 2=0.993,乙脑患者血清样品原液及倍比稀释度的病毒拷贝数分别为1184130.27、120912.86、10736.84、1053.45、121.25 copies/μL。结论本研究成功构建了检测JEV的质粒标准品,通过RT-qPCR发现其具有稳定性好、灵敏度高等优点,可应用于JEV拷贝数的定量检测。

Objective To construct a plasmid standard for detecting the copy number of Japanese encephalitis virus GⅢ(JEV),which can be used to detect JEV quantitatively.Methods The relative conserved domain sequence of JEV(prM structural protein)was amplified by reverse transcriptional polymerase chain reaction with designed primers,and the target fragment was cloned into pMD19-T vector,thus JEV plasmid standard was obtained.Then,the standard curve equation of the plasmid standard was obtained by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)and the virus copy number in the serum of JEV patients was detected.Results The RT-qPCR results of the constructed JEV plasmid standard indicated that the discrepancy of Ct value between multiple pores was small(<0.5)and the peak value of melting curve was single.The standard curve equation was y=-0.2889 x+11.516,R 2=0.993.The virus copy numbers of serum samples and their double dilution of JEV patients were 1184130.27,120912.86,10736.84,1053.45,121.25 copies/μL,respectively.Conclusion The plasmid standard for JEV detection was successfully constructed,and RT-qPCR found it to have the advantages of good stability and high sensitivity,which could be applied to the quantitative detection of JEV copy number.