烟粉虱体内木尔坦棉花曲叶病毒的LAMP快速检测技术

Detection of Cotton leaf curl Multan virus in the adult body of the whitefly Bemisia tabaci using LAMP technique

木尔坦棉花曲叶病毒(Cotton leaf curl Multan virus,CLCu Mu V)是引起世界范围内棉花曲叶病流行的主要病原之一,目前已入侵我国广东、广西、海南、福建、云南等地区。该病害由烟粉虱传播,随着烟粉虱扩散范围增加、危害不断加重,棉花曲叶病对我国棉花生产的潜在危害也日益增加。加强该病毒传播介体携带病毒的快速、特异性检测技术研发,对该病害的有效检疫与防控具有重要意义。本研究基于木尔坦棉花曲叶病毒(CLCu Mu V)的基因序列设计出LAMP检测4条特异性引物,建立LAMP扩增体系并优化扩增条件,扩增试验证明引物组合的特异性高,LAMP检测所需时间短,仅29 min即可定性检测CLCu Mu V扩增产物;该方法较普通PCR的扩增灵敏度高,可用于检测1头烟粉虱体内是否携带CLCu Mu V,简便易行,只通过颜色变绿或浊度变浑浊即可定性判断。建立的LAMP检测技术可被用于苗木上烟粉虱携带CLCu Mu V的早期检测,为介体昆虫带毒的检疫监测提供一种快速、准确和易操作的新技术。

Cotton leaf curl Multan virus（ CLCu Mu V）,transmitting by the silver leaf whitefly complex of Bemisia tabaci,has caused cotton curve leaf disease in the regions of Pakistan and India since 1997. The infested Hibiscus rose-sinensis and other host plants in the Hibisceae tribe of the family Malvaceae,were found in the region of South China. The disease was predicted to pose potential threat to cotton production in China once the whitefly complex as its vector disperse more widely. It is important and necessary to develop a quick,practicable tool for detecting CLCu Mu V presence in the whitefly body to efficiently prevent the virus from spread. Based on the conservative sequence of CLCu Mu V,4 primers specific to CLCu Mu V were designed and used in detection of the Begomovirus in a body of whitefly adult using Loop-Mediated Isothermal Amplification（ LAMP）. The reaction system for CLCu Mu V detection in the infested whitely body was set up and optimized. The LAMP technique was used to determine CLCu Mu V incident in a whitefly body within a short time of 29 min and the turbidity and green color could be oserved as indicators for CLCu Mu V present in the whitefly body. The consequences showed the tool is of specific and sensitive for CLCu Mu V detection in the whitefly.