鲤春病毒血症病毒SYBR GreenⅠ荧光定量RT-PCR方法的建立

Develompment of a SYBR GreenⅠ based real-time RT-PCR assay for rapid diagnosis of spring viremia of carp virus

为建立鲤春病毒血症病毒(SVCV)的荧光定量RT-PCR检测方法,本研究根据Gen Bank中登录的SVCV N蛋白基因的保守性序列设计并合成特异性引物,建立了该病毒的SYBR Green I荧光定量RT-PCR检测方法。结果表明,以重组质粒标准品构建的标准曲线的相关系数为0.99,在102拷贝/μL^109拷贝/μL范围内具有良好的线性关系。该方法对传染性脾肾坏死病毒、草鱼呼肠孤病毒、传染性皮下及组织坏死病毒和白斑综合征病毒其他水生动物病毒核酸扩增结果均为阴性,特异性强;该方法对SVCV检测下限为100.76 TCID50/m L,比常规PCR灵敏100倍;批内和批间的变异系数均小于7.6%,重复性好。本研究建立的荧光定量RT-PCR方法灵敏度高、特异性强、重复性好,对SVCV的快速检测及定量检测具有重要意义。

In this study, a SYBR Green I based real-time RT-PCR assay was established for detecting spring viremia of carp virus （SVCV） with a pair of primers designed according to the N sequence of SVCV. The standard cure showed that the correlation coefficient of the assay was 0.99 with a linear relationship from 102 copies/μL to 108 copies/μL of the standard recombinant plasmid. The assay was specific for detecting SVCV with a limit detection of 10-76 TCIDs0/mL which was 100 times to the regular PCR method, and had no amplifications for any other related virus DNAs. In addition, the coefficient of variation was less than 7.6% for both intra- and inter-assays. The assay established in this study is considered to be an effective tool for the rapid detection and quantification of the infection of SVCV.