博尔纳病病毒荧光定量套式RT-PCR检测方法的建立

Detection of Borna disease virus by fluorescence quantitative nested RT-PCR

目的 :建立博尔纳病病毒 (BDV)荧光定量套式RT -PCR检测方法 ,为快速、准确地定量检测BDV奠定基础。方法 :根据BDVP2 4基因序列 ,设计并合成引物和荧光标记探针。将PCR扩增的BDVP2 4基因片段克隆入载体 ,重组质粒经筛选、鉴定后 ,作为阳性模板 ,用于标准曲线的制定和样品检测。结果 :应用量组质粒制作的定量曲线循环阈值与模板具有良好的线性关系 ,相关系数为 0 .998,建立了检测BDV的荧光定量套式RT -PCR方法。检测 5 8例神经精神病患者及 5 0例健康人外周血单核细胞 (PBMC)中BDVP2 4基因片段 ,3例精神分裂症及l例帕金森病患者呈阳性 ,其余均为阴性。结论 :荧光定量套式RT -PCR方法能够避免PCR后处理导致的假阳性污染 ,并实现准确定量 ,对于研究BDV感染与人类神经精神疾病的关系有很好的应用价值。

Objective:To establish fluorescence quantitative nested RT-PCR method for detecting Borna disease virus (BDV).Methods:According to the specific sequence of BDV P24 genes,the primers and the fluorescence probe were designed and synthesized.The fragment generated by PCR was cloned into the pMD18-T vector.The positive recombinant plasmid would be used as standard quantitative template to make the standard curve for sample detection.Results:The standard curve indicated the linear relationship between Ct(cycle threshold) and template concentration (r=0.998).The fluorescence quantitative nested RT-PCR method for detection of BDV p24 fragment was established.And it was used to detect BDV p24 fragment in peripheral blood mononuclear cells(PBMC)from 58 patients with neuropsychiatric disorders and 50 healthy blood donors.4 patients with neuropsychiatric disorders were positive,and normal control negative.Conclusion:The fluorescence quantitative nested RT-PCR method for detection of Borna disease virus can eliminate PCR cross-contamination which causes false positive,and real-time detection ensures accurate quantity.It can be used to study the association between BDV infection and human neuropsychiatric disorders.