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为了建立和应用可检测基因Ⅰ型草鱼呼肠孤病毒(GCRV)的荧光定量检测方法,实验根据基因Ⅰ型GCRV的S6保守序列,分别设计扩增目的条带661 bp普通引物(P1、P2)、扩增目的条带159 bp荧光定量引物(F1、F2)和一条探针;同时构建含有目的片段的PVAX1-S6作为标准质粒,10倍梯度稀释构建质粒拷贝数与Ct值的标准曲线(Y=-3.389X+38.076)。结果表明,此方法在3.9×107~3.9×101拷贝/μL之间相关性较好,R2为0.992,最小检测量为4拷贝/μL;且与基因Ⅱ型、Ⅲ型GCRV以及其他病原核酸无交叉反应。运用该方法检测16份草鱼出血病疑似样品,阳性结果为4份;而普通PCR检测仅2份显示阳性。本研究建立了针对基因Ⅰ型GCRV的荧光定量检测方法,具有高效、特异、灵敏、可重复性强的优点,适合于目前基因Ⅰ型GCRV的临床快速检测和病毒定量分析。
In order to establish and apply fluorescent quantitative detection method of detectable gene type Ⅰ reovirus (GCRV), we designed 661 bp common primers (P1 and P2) according to the conserved sequence of genotype Ⅰ GCRV, (F1, F2) and a single probe were amplified by PCR, and PVAX1-S6 containing the target fragment was constructed as a standard plasmid. The standard curve of plasmid copy number and Ct value (Y = -3.389X + 38.076). The results showed that the correlation between this method and 3.9 × 107 ~ 3.9 × 101 copies / μL was good, R2 was 0.992, and the minimum detection was 4 copies / μL. The results showed that this method was compatible with genotypes Ⅱ and Ⅲ of GCRV and other pathogenic nucleic acids Cross reaction. Using this method to detect 16 samples of suspected grass carp hemorrhagic disease, the positive result was 4, while only 2 of the common PCR tests showed positive. This study established a fluorescence quantitative detection method for genotype Ⅰ GCRV, which has the advantages of high efficiency, specificity, sensitivity and repeatability. It is suitable for the rapid clinical detection and quantitative analysis of the genotype Ⅰ GCRV.