猪圆环病毒2型Taqman探针法实时荧光定量PCR检测方法的建立

猪圆环病毒2型（PCV2）是一种引起仔猪Sus scrofa多系统衰竭综合征、免疫抑制、皮肤肾病综合征等疾病的病原体。根据GenBank中PCV2 ORF1基因序列设计合成特异性引物和探针，以PCV2基因组DNA为模板，通过聚合酶链式反应（PCR）扩增目的片段，并将目的片段克隆至pMD18-T载体，构建阳性标准品。以阳性标准品为模板，优化荧光定量PCR反应条件，建立标准曲线，进行灵敏性、重复性和特异性验证，并应用此方法对临床样品进行检测。结果表明:阳性质粒标准品构建成功，该检测方法的引物与探针的最佳浓度皆为0.2 μmol·L-1，线性检测范围为2.1×1013~2.1×106拷贝·L-1，最低检测限值为8.828×106拷贝·L-1，且与其他相关疾病无交叉反应；各批次检测变异系数低，检测方法稳定性好。本研究建立的敏感性高、特异性好的实时荧光定量PCR方法，为PCV2的快速检测提供了新工具。

Development of Taqman real time fluorescent quantitative PCR for porcine circovirus type 2

Porcine circovirus type 2 (PCV2) is a pathogen causing postweaning multisystemic wasting syndrome (PMWS), immune suppression, and skin nephrotic syndrome of piglets. To construct specific method of detecting PCV2 nucleic acid basing on Taqman probes, PCV2 genomic DNA was used as a template for polymerase chain reaction (PCR). The specific primers of PCV2, open reading frame 1 (ORF1), and probes were designed and synthesized according to the gene sequence in GenBank. Then, the target fragment was amplified and cloned into a pMD18-T vector. The cyclic conditions of quantitative PCR were optimized by a template with a positive plasmid standard. The standard curve of real time fluorescent quantitative PCR for PCV2 was established and the sensitivity, repeatability, and specificity of the method were verified by three repeatability tests. Results showed that a positive plasmid was successfully constructed. The optimal concentration of primers and probe was 0.2 μmol·L-1, and the minimum detection threshold was 8.828×106 copies·L-1. The linear range of detection was 1013-106 copies·L-1. This method had no cross reaction of related diseases, a low coefficient of variation, and well repeatability between testing of every batch. From this study then, the high sensitivity and specific real-time quantitative PCR for PCV2 could be used as a tool for rapidly diagnosing PCV2 infectious diseases.