猪源TTV+Taqman实时荧光定量PCR检测方法的建立和应用.pdfVIP

上海市畜牧兽医学会 2011 年学术年会论文集 猪源 TTV Taqman 实时荧光定量 PCR 检测 方法的建立和应用 侯军委，周艳君，王礞礞，李国新，于 海，童光志 （中国农业科学院上海兽医研究所，上海200241 ） 摘 要：根据TTV1和TTV2的非编码区（UTR）的保守序列分别设计并合成两套特异性引物和Taqman 探针， 建立了鉴别TTV1和TTV2 的Taqman实时荧光定量PCR方法。通过常规PCR 方法分别克隆TTV1和TTV2的非 编码区（UTR）的保守序列并将其连入 pMD-18T 载体，制备阳性标准品，优化反应条件，以 10 倍系列稀释 的标准品分别绘制标准曲线，TTV1 标准曲线的相关系数为 0.984，TTV2 标准曲线的相关系数为 0.994。检 测结果显示，两种方法的灵敏度均可达10copies/μL，除猪源TTV外，对猪繁殖与呼吸综合征病毒、猪瘟 病毒、猪2型圆环病毒、猪流感病毒检测结果均为阴性。该方法重复性好，批内和批间变异系数均小于3%。 检测采集自广西和内蒙古的44份病料，TTV1的阳性率为47.73%，TTV2阳性率为70.45%，TTV1和TTV2 混 合感染的阳性率为31.82%。猪源TTV检测方法的建立为该病毒的流行病学调查和定量提供了有效的手段。 关键词：TTV1 ； TTV2 ；Taqman实时荧光定量PCR DEVELOPMENT AND APPLICATION OF TAQMAN REAL-TIME FLUORESCENCE QUANTITATIVE PCR ASSAY FOR DETECTION OF PORCINE TORQUE TENO VIRUS HOU Jun-wei, ZHOU Yan-jun, WANG Meng-meng, LI Guo-xin, YU Hai, TONG Guang-zhi* (Division of Swine Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China) Abstract ：Two Taqman real-time fluorescence quantitative PCRs were established using two sets of primers and probes based on the conserved untranslated region (UTR) of porcine Torque Teno virus genotype 1 and 2 (TTV1 and TTV2). The UTR sequences of TTV1 and TTV2 were cloned into the pMD-18T vector and a series of diluted recombinant plasmids were used to generate standard curves. The quantitative PCR assays could detect as less as 10 copies of target DNA of TTV1 or TTV2 . The assay showed good specificity and did not cross react with other porcine viruses (PRRSV, CSFV, PCV2 and SIV). The variation coefficient of intra-batch or inter-batch were both below 3% , which indicated good reproducibility. 44 clinical samples of pigs were detected by the two quantitative