香蕉花叶心腐病的分子诊断研究初报

MOLECULAR DIAGNOSIS OF BANANA M0SAIC AND HEART ROT DISEASE

用核酸杂交和聚合酶链式反应（ＰＣＲ），对黄瓜花叶病毒（ＣＭＶ）引起的香蕉花叶心腐病进行了诊断检测研究。实验结果表明，用（３２）Ｐ标记的克隆化的ＣＭＶ－ＣＰ基因ｃＤＮＡ探针，通过斑点杂交法可检测出１０ｐｇ的病毒ＲＮＡ，并能够从１００ｎｇ植物总核酸中检测到病毒核酸，对感染ＣＭＶ的香蕉植株可做出正确诊断；用ＣＭＶ－ＣＰ基因两端引物，通过反转录——聚合酶链反应（ＲＴ—ＰＣＲ）．对染病香蕉植株总核酸直接扩增，可获得异性的ＤＮＡ片断。

Molecular diagnosis techniques,focused on nucleic acid hybridization and polymerase chainreaction(PCR),were applied in the detecting studies of cucumher mosaic (CMV),thecausative agent of banana mosaic and heart rot disease。Preliminary results indicated that dot blothybridization assay,using (32)p-labelled cDNA probe derived from recombinant plasmid pBV18containing CMVCP gene insert,could be a specific and sensitive method for virus detection ofbanana,Using this method 10 pg RNA could be detected from purified virion,and the minimurndetecting level of concentrated total nucleicacid of diseased banana leaves was 100 ng。It wasfound that direct blotting of crude sample extracted from infected plant could alsok give hybridization spots while those from heaIthy ones gave negative results,Employing a pair of primers ofCMV CP gene,the amplified products specific to CMV CP gene verified by Southern blottingtest,were obtained by RT-pCR from total RNA of diseased banana as well as purified RNA ofvirion.