| 猪源TTV Taqman实时荧光定量PCR检测方法的建立和应用 |
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| 引用本文： | 侯军委,周艳君,王礞礞,李国新,于海,童光志.猪源TTV Taqman实时荧光定量PCR检测方法的建立和应用[J].中国兽医寄生虫病,2011,19(2):13-19. |
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| 作者姓名： | 侯军委 周艳君 王礞礞 李国新 于海 童光志 |
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| 作者单位： | 中国农业科学院上海兽医研究所,上海,200241 |
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| 基金项目： | 上海市科技人才计划项目，国家生猪现代产业技术体系建设项目 |
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| 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pMD18-T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10 copies/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。
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| 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 |
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| HOU Jun-wei,ZHOU Yan-jun,WANG Meng-meng,LI Guo-xin,YU Hai,TONG Guang-zhi.DEVELOPMENT AND APPLICATION OF TAQMAN REAL-TIME FLUORESCENCE QUANTITATIVE PCR ASSAY FOR DETECTION OF PORCINE TORQUE TENO VIRUS[J].Chinese Journal of Veterinary Parasitology,2011,19(2):13-19. |
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| Authors: | HOU Jun-wei ZHOU Yan-jun WANG Meng-meng LI Guo-xin YU Hai TONG Guang-zhi |
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| Institution: | HOU Jun-wei,ZHOU Yan-jun,WANG Meng-meng,LI Guo-xin,YU Hai,TONG Guang-zhi(Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China) |
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| 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 pMD18-T 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 from Neimeng and Guangxi were detected by the two quantitative PCR assays,and the results showed that 47.73%,70.45% and 31.82% of the sample were positive for TTV1,TTV2,and both,respectively. |
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| Keywords: | TTV1 TTV2 Taqman real-time fluorescence quantitative PCR |
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