猪细环病毒数字PCR定量检测方法的建立

Development of a Digital PCR Assay for Quantitative Detection of Torque Teno Sus Virus

[目的]实现猪细环病毒(TTSuV)的准确定量检测。[方法 ]根据TTSuV的序列特点,设计特异性引物、探针,建立数字PCR检测技术。对数字PCR反应体系中的引物和探针浓度进行优化,分析方法的灵敏度、特异性,并初步应用于进行临床检测。[结果 ]最终确定TTSuV1a和TTSuV1b数字PCR反应体系中最佳引物浓度均为250 nmol/L,最佳探针浓度均为300 nmol/L,TTSuV1a型和TTSuV1b型灵敏度均可达到单个拷贝数;以猪圆环病毒Ⅱ型、猪细小病毒和猪伪狂犬病毒进行特异性试验,结果无交叉反应;批内和批间试验表明,该方法的重复性良好;本实验室留存的92份血清样本的检测结果与其背景信息一致。[结论 ]本研究建立的TTSuV数字PCR法具有特异性强、灵敏度高、检测限低等优点,可用于TTSuV的定量检测。

[Objective] In order to accurately and quantitatively detect the Torque Teno Sus virus （TTSuV） . [Methods] Specific primers and probes were designed and synthesized based on the sequence of TTSuV, then a digital PCR detection assay （dPCR） was established. After optimizing the working concentration of primers and probes, the sensitivity and specificity of the established reaction system were tested, and preliminary application was carried out. [Results] The optimal concentration of primers for both TTSuVla and TTSuVlb genes had the same value of 250 nmol/L, and the optimal concentration of probes for the two genes was 300 nmol/L. As to the sensitivity of dPCR method, results showed single copy of TTSuVla and TTSuVlb could be detected. Then its specificitywas evaluated by detecting TTSuV, Parvovirus （PPV）, Porcine circovirus type 2 （PCV-2） and Pseudorabies virus （PRV） simultaneously, and no cross reaction was observed. Furthermore, good repeatability of the method was confirmed by intra and inter assay. At last, 92 clinical serum samples derived from our laboratory were conducted detection, results of which showed good consistence with their background information. [Conclusion]The established dPCR method was specific, sensitive and repeatable., and it could be used in quantitative detection of TTSuV.