利用PCR方法检测锦鲤疱疹病毒3个主要靶基因

Multiple gene detection of koi herpesvirus by PCR

为建立锦鲤疱疹病毒(KHV)多靶基因PCR检测方法,本实验将KHV接种鲤鱼鳍条细胞,收获病变细胞悬液,提取DNA,根据GenBank中登录的KHV基因序列及出入境检验检疫行业标准推荐的基因(ORF7),设计合成3对特异性引物,针对胸苷激酶基因(TK)、聚合酶基因(Sph)和ORF7基因进行PCR检测。通过优化后的反应体系进行特异性、敏感性试验和样品检测。结果表明:3对引物能够分别特异性扩增出409bp、292bp和484bp片段;敏感性试验表明对TK基因检测的敏感性高于Sph和ORF7基因,其最低检测量为1.9×106copies/μL;采用优化的3个PCR方法对8个有临床症状的样品进行检测,其中3份样品的3个基因PCR扩增结果均为阳性。因此,本研究选取的3个基因均可用于KHV的检测及确证实验。

To exploratory the feasibility of multiple gene detection of koi herpesvirus （KHV） by PCR, the PCR detections were conducted to amplify the TK and Sph genes of KHV to comparing with the amplification of ORF7 which was recommended by inspection and quarantine standard for KHV detection. The three genes were amplified with the spcefic primers and the the template DNA of virus extracted from the KHV infected KF cells. The results showed that the fragments of 409 bp （TK）, 292 bp （Sph）, and 484 bp （ORF7） could be specifically amplified with a detection limit at 1.9×106 copies/μL of KHV. It was evident that the all of these gene could be used to detection of KHV.