一种基于RPA的番茄褪绿病毒检测方法

Detection of Tomato chlorotic virus based on RPA

番茄褪绿病毒Tomato chlorosis virus(ToCV)引起番茄褪绿病毒病,给番茄生产造成严重危害。开发快速准确的检测方法对该病害的防控具有重要意义。利用番茄褪绿病毒外壳蛋白(CP)基因序列,设计特异性引物,建立了ToCV的重组酶聚合酶等温扩增(recombinase polymerase amplification,RPA)检测方法,同时分析了该方法的灵敏度和特异性。结果表明,建立的ToCV-RPA方法在38℃恒温下40 min可从ToCV阳性的番茄样品中扩增出246 bp的特异性条带。扩增时间短,对设备要求低,且与番茄其他病毒无交叉反应,特异性好,灵敏度可达到PCR方法的10倍,适用于ToCV的快速检测。

Tomato chlorosis virus(ToCV)is the pathogen of tomato chlorosis virus disease,which causes serious damage to tomato production.It is important to develop rapid and accurate detection methods for the prevention and control of tomato chlorosis virus disease.Based on the CP gene sequence of ToCV,the specific primers were designed for the detection of virus via recombinase polymerase amplification(RPA).The specificity and sensitivity of the method were evaluated.The result showed that 246 bp specific band from ToCV-positive tomato samples could be amplified by the established ToCV-RPA method.The reaction condition was 38℃for 40 min.The method has advantages of short operation time,low equipment requirement,and good specificity and there was no cross-reaction with other tomato viruses.The sensitivity was 10 times higher than that of the PCR method.It was suitable for rapid detection of ToCV.