应用RT-PCR法快速检测马铃薯卷叶病毒

A rapid method for detection of potato leafroll virus by reverse transcription polymerase chain reaction amplification

根据马铃薯卷叶病毒的外壳蛋白基因序列 ,设计合成了一对寡核苷酸引物 ,从感染PLRV的马铃薯病叶组织中提取出病毒的RNA ,进行cDNA合成及PCR扩增 ,得到一条长度约 6 2 0bp的特异PCR扩增产物 ,与理论设计的外壳蛋白基因大小一致。建立了快速灵敏简便的PLRV检测的新方法 ,其灵敏度要比PLRV的血清学检测方法高 10 4倍 ,在基因水平上为PLRV的检测提供了更新手段 。

A pair of DNA primers were designed and synthesized based on the nucleotide sequence of the coat protein(CP) gene of potato leafroll luteovirus (PLRV). The PLRV RNA was directly extracted from virus-infected potato leaves, and utilized for cDNA synthesizing, then it was amplified by polymerase chain reaction (PCR). A specific PCR fragments about 620 bp thus was obtained ,and it was the expected length of PLRV CP gene. The expriment provides a rapid, sensitive and relatively inexpensive means of PLRV detection and identification, and the RT PCR assay for PLRV is about 10 4 fold more sensitive than Enzyme linked Immunosorbent Assay (ELISA). It also provides a new method that can take the place of detection and identification of PLRA in gene level and that has been applied by the Center of Potato Detection of the Plant Quarantine Institute of Ministry of Agriculture