Blaser Simon, Diem Hanspeter, von Felten Andreas, Gueuning Morgan, Andreou Michael, Boonham Neil, Tomlinson Jennifer, Müller Pie, Utzinger Jürg, Frey Beatrice, Frey Jürg E, Bühlmann Andreas
Department of Method Development and Analytics, Agroscope; Swiss Tropical and Public Health Institute; University of Basel;
Swiss Federal Plant Protection Service, Federal Office for Agriculture.
J Vis Exp. 2018 Oct 29(140):58502. doi: 10.3791/58502.
The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many countries around the world. Severe yield losses are caused by direct feeding, and even more importantly, also by the transmission of more than 100 harmful plant pathogenic viruses. As for other invasive pests, increased international trade facilitates the dispersal of B. tabaci to areas beyond its native range. Inspections of plant import products at points of entry such as seaports and airports are, therefore, seen as an important prevention measure. However, this last line of defense against pest invasions is only effective if rapid identification methods for suspicious insect specimens are readily available. Because the morphological differentiation between the regulated B. tabaci and close relatives without quarantine status is difficult for non-taxonomists, a rapid molecular identification assay based on the loop-mediated isothermal amplification (LAMP) technology has been developed. This publication reports the detailed protocol of the novel assay describing rapid DNA extraction, set-up of the LAMP reaction, as well as interpretation of its read-out, which allows identifying B. tabaci specimens within one hour. Compared to existing protocols for the detection of specific B. tabaci biotypes, the developed method targets the whole B. tabaci species complex in one assay. Moreover the assay is designed to be applied on-site by plant health inspectors with minimal laboratory training directly at points of entry. Thorough validation performed under laboratory and on-site conditions demonstrates that the reported LAMP assay is a rapid and reliable identification tool, improving the management of B. tabaci.
烟粉虱Bemisia tabaci（Gennadius）是一种极具重要性的入侵害虫，影响着世界上许多国家的蔬菜和观赏作物生产。直接取食会导致严重的产量损失，更重要的是，它还会传播100多种有害植物病原病毒。与其他入侵害虫一样，国际贸易的增加促进了烟粉虱向其原生范围以外地区的扩散。因此，在海港和机场等入境点对植物进口产品进行检查被视为一项重要的预防措施。然而，只有当可疑昆虫标本的快速鉴定方法随时可用时，这种针对害虫入侵的最后一道防线才有效。由于对于非分类学家来说，区分受监管的烟粉虱与其无检疫地位的近缘种在形态上很困难，因此已开发出一种基于环介导等温扩增（LAMP）技术的快速分子鉴定方法。本出版物报告了该新方法的详细方案，描述了快速DNA提取、LAMP反应的设置及其结果解读，可在一小时内鉴定烟粉虱标本。与现有的检测特定烟粉虱生物型的方法相比，所开发的方法在一次检测中针对整个烟粉虱物种复合体。此外，该方法设计为由植物卫生检查员在入境点直接进行现场应用，只需极少的实验室培训。在实验室和现场条件下进行的全面验证表明，所报道的LAMP检测方法是一种快速可靠的鉴定工具，可改善对烟粉虱的管理。
