猪细环病毒实时荧光PCR检测技术的建立及应用

Development and application of real-time PCR assays for detection of the Torque teno sus virus

为了实现猪细环病毒(TTSuV)快速检测,给TTSuV的调查、监测和防控提供可行性、操作性强的技术支持。本试验根据TTSuV的序列特点设计特异性引物、探针,应用实时荧光PCR技术检测TTSuV1a型和TTSuV1a1b型。以猪细小病毒、猪圆环病毒Ⅱ型和猪伪狂犬病毒进行特异性试验,无交叉反应。该检测方法具有较好的特异性和重复性,TTSuV1a灵敏度可达9.2拷贝/μL,TTSuV1b灵敏度可达5.6×101拷贝/μL;批内变异系数均小于1%,批间变异系数均小于3%。将该方法与PCR法、PCR-DHPLC进行比较,结果证实该方法具有较好的准确性。对92份血清样本进行检测,发现有58份TTSuV1a阳性、62份TTSuV1b阳性、51份TTSuV1a和TTSuV1b共感染阳性。本研究建立的TTSuV实时荧光PCR法具有特异、敏感、快速、重复性好等优点,可用于TTSuV感染的分子流行病学调查。

To identify the Torque teno sus virus（TTSuV）,real-time PCR assay was performed in this study. Specific primers for the partial region of the TTSuVla and TTSuVlb were selected to conduct the real-time PCR assays. The specificity test was performed with PPV, there was not cross reaction between PCV Ⅱ and PRV, and better specificity and repeatability were abserved. Sensitivity analysis showed that the developed real-time PCR for TTSuVla could detect 9.2 cop- ies/μL,and 5. 6 × 101 copies/μL for TTSuVlb. The coefficient of variation in a group was less than l~,the coefficient of variation between the groups was less than 3%,with better accuracy compared with the PCR,PCR-DHPLC. To detect the serum samples, 58 TTSuVla positives re- suits could be observed by real-time PCR for 92 samples,62 TTSuVlb positives,51 TTSuVla and TTSuVlb coinfected positives. The method with good specification, sensitivity, repeatability, and quickness could be used for epidemiological investigation of TTSuV.