棉花皱叶病毒LAMP检测方法的建立

Detection of Cotton leaf crumple virus by Loop-mediated Isothermal Amplification

棉花皱叶病毒(Cotton leaf crumple virus,CLCrV)是危害棉花的重要病毒之一,属中国进境检疫性植物病毒,本研究根据其DNA-B基因序列设计LAMP特异性引物,通过试验建立CLCrV的LAMP检测方法。该方法检测快速,15min即可检测到扩增曲线;特异性强,可有效区分同属的CLCrV、棉花曲叶病毒(CLCuV)、非洲木薯花叶病毒(ACMV)及番茄黄化曲叶病毒(TYLCV);灵敏度高,可检测到约43fg/μL的总DNA,比普通PCR灵敏度高1 000倍;并可通过肉眼观察浊度或颜色反应进行结果判断。该方法适用于现场CLCrV的快速检测。

Cotton leaf crumple virus （CLCrV） is one of the most important pathogens of cottons and is one of the entry quarantine plant virus of China. For developing a rapid and sensitive detection tech- nique to detect CLCrV in order to prevent its introduction and spread, we have established loop-medi- ated isotermal amplification （LAMP） method, which using DNA-B fragment sequence as the target, after the optimization of primers and reaction conditions. The sensitivity of the method was about 43 fg/μL of total DNA, which was 1 000 times higher than that of the conventional PCR. The method has good specificity and can effectively distinguish four geminiviruses, CLCrV, Cotton leaf curl virus （CLCuV）, African cassava mosaic virus （ACMV）, and Tomato yellow leaf curl virus （TYLCV）. De- tection results can be judged by visual observation of turbidity or color reaction and not require com- plex equipment. The method can be used for the rapid detection of the filed CLCrV.