犬细小病毒荧光定量PCR检测方法的建立

Establishment of SYBR-Green Real-time PCR for Canine Parvovirus Detection

建立一种敏感性高、重复性好、特异性强的犬细小病毒荧光定量PCR检测方法。根据GenBank中犬细小病毒(CPV)VP2蛋白基因序列,选择其保守序列设计引物并构建质粒,对系列稀释的质粒进行扩增构建标准曲线,其相关系数为0.997,所建立的方法最低可检测10拷贝/μL,且对猪细小病毒、猪圆环病毒没有阳性检出,进行5次重复检测,体系的变异系数(CV)<2%,另对12份犬细小病毒阳性血液样品进行检测,均得到209kb大小的片段。表明所建立的SYBR-Green荧光定量PCR方法具有良好特异性、敏感性和重复性,可用于犬细小病病毒的病原检测。

A real-time PCR method was established to detect CPV, which has sensibility, specificity and repetitiveness. Primers were designed according to VP2 protein gene of CPV-2, and the replieon was inserted into pSURE-T vector. The plasmids were diluted serialy and amplificated to establish the standard curves （R2 =0. 997）, the lowest concentration, which can be detected, is 10 copies/~L. Both procine par- vovirus （PPV） and procine circovirus （PCV） were negative groups. The samples were detected five times with CV〈2% ,and the 12 CPV positive blood samples were detected by the method, the 209 kb gel fragments were obtained. All results showed that the SYBR Green real time PCR established has better specificity, sensibility and repetitiveness.