IC-RT-PCR检测葡萄卷叶病毒Ⅲ的研究

Study on IC-RT-PCR for Detecting Grapevine Leaf Roll-associated Virus-3

结合ELISA和反转录聚合酶链式反应(RT-PCR)的特点,引入了另外一种方法,即免疫捕捉反转录聚合酶链式反应(IC-RT-PCR)对葡萄卷叶病毒Ⅲ(GLRaV-3)进行了检测。从免疫捕捉病毒、释放RNA到反转录成cDNA,最后扩增出了约300bp的预期目的片段。为了进一步明确PCR扩增的特异性,将PCR产物连接到pGEM-T载体上,转化大肠杆菌E.coliJM109菌株,通过蓝白斑筛选,获得重组质粒,提取质粒DNA,经酶切对重组质粒进行鉴定,结果得到了含有目的片段的重组质粒。由此证明,IC-RT-PCR检测GLRaV-3结果准确可靠。最重要的是它弥补了ELISA易出现假阴性、假阳性以及RT-PCR提取RNA难等的缺点,不失为病毒检测的新型方法。

The authors detected GLRaV- 3 by immunocapture reverse transcriptase polymerase chain reaction（ IC - RT - PCR） through the following steps from capturing virus, releasing RNA, transcribing cDNA to producing the target fragments of about 300bp. To check the specificity and efficiency of GLRaV- 3 cDNA fragments amplified, pGEM - T jointing the PCR products was used to transform E. coli JM109 and the recombinant plasmids were obtained by blue plaque screening. Further characterization by restriction enzyme digestion and PCR showed that the plasmids contained the target fragments, indicating that the technique of IC - RT - PCR was reliable.