摘要
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.
出处
《贵州农业科学》
CAS
北大核心
2014年第6期104-108,共5页
Guizhou Agricultural Sciences
基金
贵州省科学技术基金项目[黔科合J字(2010)2260]
毕节市科技局项目"贵州黑山羊羔羊口疮病防治技术攻关"[毕科合字(2012)24号]
重庆市基本科研业务费专项"羊口疮病原分离鉴定及生物学特性研究"(11618)
关键词
羊口疮病毒
检测方法
PCR
ORFV
real-time fluorescence quantification PCR
detection method