SYBR-GreenⅠ实时荧光PCR检测传染性鲑鱼贫血病

DETECTION OF INFECTIOUS SALMON ANAEMIA VIRUS WITH SYBR-GREEN I REAL-TIME PCR ASSAY

[目的]利用SYBR-GreenI实时荧光PCR检测传染性鲑鱼贫血病。[方法]根据传染性鲑鱼贫血病毒(ISAV)Y10404的序列合成部分基因片段,将其插入到T载体中,构建阳性质控品;并设计合成一对特异性引物。利用阳性质控品建立SYBR-GreenⅠ实时荧光PCR方法,对反应体系进行优化,进行特异性和敏感性分析,并制作标准曲线。[结果]最佳引物终浓度为0.5μmol/L。所建立的SYBR-GreenⅠ实时荧光PCR方法能够特异地检出ISAV;最低检出浓度为9.5×10-8μg/μL,其灵敏度比传统PCR高1000倍;能够从阳性样品中检出ISAV,Tm值为82.5℃。建立了标准曲线,其r2>0.99。

According to the sequence of the accession No.Y10404 of infectious salmon anaemia virus （ISAV）, a partial gene fragment was synthesized and inserted into T vector to establish positive quality control and a pair of specific primers was designed and synthesized. The SYBR-Green I real-time PCR was established by the positive quality control. The reactive system was optimized, its specificity and sensitivity were analyzed and the standard curve was performed. The established assay was applied into the detection of the positive sample. The results showed that the optimized primers＇ concentration was 0.5urnol/L. The assay could detect ISAV specifically and was 1000 fold more sensitive than conventional PCR. And its sensitivity was 9.5× 10^-8ug/uL. ISAV could be detected fi＇om the positive sample, and Tm value was 82.5℃. A standard curve was obtained and had its r^2〉0.99 and was highly reliable, so it can be applied into quantitative detection.