实时荧光定量TaqMan-PCR检测小鼠多瘤病毒方法的建立

目的 建立一种快速、特异、敏感的小鼠多瘤病毒 (murine polyomavirus, MPyV)荧光定量 TaqMan PCR检测方法, 用于MPyV核酸的检测。 方法 根据GenBank中小鼠多瘤病毒基因组序列设计了一对特异性引物及TaqMan探针, 扩增长度为69 bp的片段, 通过优化反应体系和反应条件, 对构建的重组质粒标准品进行检测, 并绘制标准曲线, 进行特异性、敏感性和重复性试验。最后用建立的方法对人工感染MPyV的肺脏、脾脏和粪便, 及86 份小鼠临床样本进行检测, 验证在临床应用中的效果。 结果 所建立的检测方法特异性强, 只能在小鼠多瘤病毒DNA中检出荧光信号, 检测灵敏度达到100拷贝, 批内和批间重复性好, 检测结果变异系数(CV)均小于1.13%, 人工感染MPyV小鼠的肺脏、脾脏和粪便均为阳性, 对86份临床样本进行检测, 有3份样本呈阳性(阳性率为3.5%)。 结论 建立的MPyV荧光定量TaqMan-PCR检测方法特异性强、敏感性高、重复性好, 适合用于MPyV临床诊断、日常监测和流行病学调查。

Establishment of TaqMan real-time fluorescent quantitative PCR for detection of Murine polyomavirus

Objective To establish a rapid, specific and sensitive TaqMan real-time fluorescence quantitative PCR assay for detection of murine polyomavirus (MPyV). Methods The specific primers and TaqMan probe were designed based on genome sequence of MPyV. The primers amplified a 69 bp fragment. After optimizing the reaction system and reaction condition, the standard curve was plotted by detecting recombinant plasmid standards. The specificity, sensitivity and reproducibility of this method were evaluated. In addition, samples of lungs, spleens and feces obtained from experimentally infected mice and 86 clinical samples were used to validate the efficacy of this real-time PCR assay. Results The specificity assay showed that this assay could specifically detect MPyV and the sensitivity for MPyV was about 100 copies/well. The coefficients of variation (CV) of both intra-assay and inter-assay were less than 1.13%. All of the samples from experimentally infected mice were positive for MPyV and 3 out of 86 clinical samples were positive by this TaqMan-PCR detection with a positive rate of 3.5%. Conclusions The real-time fluorescence quantitative TaqMan-PCR assay established in this study has high specificity, sensitivity and stability. It can be used for clinical diagnosis, routine detection and epidemiological investigation of murine polyomavirus infections.