猪圆环病毒2型和猪细小病毒双重PCR 检测方法的建立及应用

摘要

To identify and differentiate rapidly the cause of clinical diseases.A duplex polymerase chain reaction was optimized to simultaneously detect two pathogens of Porcine Circovirus 2 ( PCV2 ) and Porcine Parvovirus (PPV) in this article.Two sets of specific primers were designed according to the sequences of PCV2 and PPV at the GenBank.By using two pairs of virus specific primers, two PCR assay were established to amplify the conservative regions of the two viruses, respectively.Consequently, a duplex PCR method to detect the two viruses in one tube was developed. The duplex PCR system would amplify a 245 bp for PCV2 and 475 bp fragment for PPV a imultaneously or separately in the samples,depending on its infection status. But not specific band amplified from other four pig pathogenic viruses.As little as 1 pg of PCV2 and 1 pg of PPV DNA were detected using gel electrophoresis in the duplex PCR.To evaluate the duplex PCR,105 clinical samples were comparatively detected.The data showed that the duplex PCR method being 100% coincidence with the single PCR, and it can be used for this two viruses detection and differential diagnosis.%根椐GenBank中登录的猪圆环病毒2型（PCV2）和猪细小病毒（PPV）核苷酸序列，分别设计2对引物，在建立各病毒单项PCR技术的基础上，优化双重PCR反应条件，建立了2种病毒的双重PCR检测方法，用这2对引物对同一样品中的PCV2和PPV核酸模板进行双重PCR扩增，结果可同时扩增PCV2的245 bp、PPV的475 bp的特异性片段，而对其他4种病原的PCR扩增结果均为阴性。敏感性测定结果表明，该双重PCR技术能检出1 pg的PCV2和1 pg的PPV模板。用105份临床病料对本研究双重PCR技术和单项PCR技术进行对比验证，结果显示，两者的总符合率为100%。表明建立的双重PCR检测方法具有特异、快速、准确的特点，可用于对这两种病毒的同时检测和鉴别诊断。