羊口疮病毒SYBR Green Ⅰ实时荧光定量PCR的建立及应用

Establishment and Application of SYBR Green I Real-time Fluorescence Quantification PCR for ORFV

为羊口疮（Orf）的分子流行病学调查、早期快速诊断及细胞培养物的检测等提供参考，根据GenBank 发表的羊口疮病毒（OrfV）B2L 基因序列设计合成1对引物，以 pMD18-T-B2L 重组质粒为阳性标准品，建立了检测 OrfV 核酸的 SYBR Green Ⅰ实时荧光定量 PCR 方法，并进行了临床应用。结果表明：建立的 SYBR Green Ⅰ实时荧光定量 PCR 的线性关系良好，标准曲线的相关系数达到-1；最低检测限为1．0x10^3 copies／μL，比常规 PCR 方法高100倍，与山羊痘和口蹄疫疫苗毒均不发生交叉反应；组内变异系数为1．061％-2．873％，组间变异系数为0．397％-3．829％。用该方法检测5例 OrfV 感染临床病例和8份不同代次的 OrfV 细胞培养物，阳性率均为100％。SYBR Green Ⅰ实时荧光定量 PCR 方法灵敏度高、特异性强、重复性好，可以用于 OrfV 的病原检测及定量分析。

The detection method of SYBR Green I real-time fluorescence quantification PCR for ORFV nucleic acid was established based on the positive standard of pMD18-T-B2L recombinant plasmid according to OPFV B2L gene sequence recommended by GenBank and the detection method was used in clinical application to provide a reference for ORFV molecular epidemiological investigation;early rapid diagnosis and detection of cell culture.The results showed that the established SYBR Green I real-time fluorescence quantification PCR has a good linear relation and the correlation coefficient of the standard curve was up to -1,and the lowest detection limit was 1.0 x 10^3 copies/μL,100 times more than the conventional PCR. Moreover,there was no cross reaction with GPV and FMDV, and the variable coefficient of intra-group and inter-group was 1.061%-2.873% and 0.397%-3.829% respectively.The positive rate of 5 ORFV clinical cases and 8 ORFV cell cultures with different generation detected by the established SYBR Green I real-time fluorescence quantification PCR was 100% all.The established SYBR Green I real-time fluorescence quantification PCR with high sensitivity, strong specificity and good repeatability can be used in detection and quantitative analysis of ORFV pathogen.