猪细环病毒PCR-DHPLC检测技术的建立及应用

Development and Application of PCR-DHPLC Assays for Detection of the Swine Torque Teno Sus Virus

为了应用PCR结合变性高效液相色谱(DHPLC)技术检测猪细环病毒1型和2型,根据猪细环病毒的序列特点设计特异性引物,PCR扩增产物经DHPLC技术进行快速检测。选择猪细小病毒、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;用质粒标准品进行TTSuV1和TTSuV2的PCR-DHPLC检测,灵敏度可达1.0×101拷贝·μL-1。对80份血清样本分别应用实时荧光PCR法、PCR-凝胶电泳法及本文所建立的PCR-DHPLC法与进行检测,发现TTSuV1有63份PCR-DHPLC阳性,63份实时荧光PCR阳性,54份PCR-凝胶电泳阳性;TTSuV2有69份PCR-DHPLC阳性,69份实时荧光PCR阳性,56份PCR-凝胶电泳阳性。本试验建立的PCR-DHPLC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 ident,ify the Torque teno sus virus （TTSuV）, a PCR-DHPLC assay was developed i in this study. Primers specific for the partial region of the TTSuV1 and TTSuV2 were selected to conduct the PCR DHPLC assays. The specificity test was performed with PPV, PCV Ⅱ and PRV and no cross reaction was found. The PCR-DHPLC for TTSuV had good specificity and nice repeatability. Sensitivity analysis showed that the developed PCR-DHPLC could detect 1.0×10^1copy·μL^-1. The established method and the Real-time PCR were used to detect 80 serum sam- ples, the results showed that 63 samples were TTSuV1 positive by PCR-DHPLC, and it was con- sistent with real-time PCR test; 69 samples were TTSuV2 positive by PCR-DHPLC, and it was consistent with real-time PCR test, 56 samples were positive by normal RT-PCR. The method showed nice specification, sensitivity, repeatability, and quickness, and it could be used in epide miological investigation of TTSuV.