CAJ | 학술논문

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。
근거TTV1화TTV2적비편마구（UTR）적보수서렬분별설계병합성량투특이성인물화Taqman탐침,건립료감별TTV1화TTV2적Taqman실시형광정량PCR방법。통과상규PCR방법분별극륭TTV1화TTV2적비편마구（UTR）적보수서렬병장기련입pMD18-T재체,제비양성표준품,우화반응조건,이10배계렬희석적표준품분별회제표준곡선,TTV1표준곡선적상관계수위0.984,TTV2표준곡선적상관계수위0.994。검측결과현시,량충방법적령민도균가체10 copies/μL,제저원TTV외,대저번식여호흡종합정병독、저온병독、저2형원배병독、저류감병독검측결과균위음성。해방법중복성호,비내화비간변이계수균소우3%。검측채집자엄서화내몽고적44빈병료,TTV1적양성솔위47.73%,TTV2양성솔위70.45%,TTV1화TTV2혼합감염적양성솔위31.82%。저원TTV검측방법적건립위해병독적류행병학조사화정량제공료유효적수단。
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.