荧光定量PCR检测土拨鼠肝炎病毒核酸方法的建立

目的建立土拨鼠肝炎病毒（woodchuck hepatitis virus,WHV）核酸的荧光定量PCR（Real-time PCR）检测方法,应用于土拨鼠肝炎病毒模型的研究。方法分别根据土拨鼠肝炎病毒核心抗原（WHcAg）和表面抗原（WHsAg）的DNA序列设计13对扩增引物,从中筛选无非特异性扩增及引物二聚体且灵敏度高的引物,用于土拨鼠血清中WHV DNA的Real-time PCR检测。建立感染土拨鼠肝炎病毒的土拨鼠血清中WHV核酸的Real-timePCR检测方法。结果根据WHsAg基因的5＇端设计的一对引物WHVSF1与WHVSR1,检测灵敏度可达1×101拷贝/μL,病毒拷贝数与Real-time PCR Ct值的标准曲线的R2值为0.997,且电泳未见明显非特异性条带及引物二聚体。结论建立了土拨鼠血清中WHV DNA的Real-time PCR检测方法,该方法为进一步研究土拨鼠肝炎病毒模型奠定了基础。

Establishment of Real-time PCR Method for Detection of Woodchuck Hepatitis Virus DNA

Objective To establish Real-time PCR method for detection of woodchuck hepatitis virus（WHV） DNA.Methods 13 pairs of primers were designed according to the coding sequence of woodchuck hepatitis virus core antigen（WHcAg） and woodchuck hepatitis virus surface antigen（WHsAg）.The primers demonstrated high sensitivity and specificity were used for Real-time PCR detection of WHV DNA in woodchuck serum.Then,this method was applied in detection of WHV DNA in serum of woodchuck.Results The selected primers pair（WHVSF1,WHVSR1） which based on 5′ end of WHsAg gene showed a detection limitation of 1×101copies/μL.The R2 value of standard curve of viral copy number and the Real-time PCR Ct value was 0.997,and no mixed bands were observed during electrophoresis.Conclusion Real-time PCR method for detection of WHV DNA in serum of WHV infected woodchuck was established,which could be used in WHV model.