Liu Shuhua, Luo Ju, Liu Rui, Zhang Chenguang, Duan Dekang, Chen Hongming, Bei Wenyong, Tang Jian
State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou,, China.
Plant Protection and Quarantine Station in Longyou, Longyou, Zhejiang Province, China.
J Econ Entomol. 2018 Dec 14;111(6):2869-2875. doi: 10.1093/jee/toy232.
The brown planthopper (BPH), Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), is a destructive rice pest of Asia. Currently, one important monitoring method of BPH is through black light trapping. However, two sibling species of BPH, Nilaparvata bakeri (Muri) and Nilaparvata muiri China, can also be trapped by black light, and these species feed only on gramineous weeds rather than on rice. Therefore, the accurate identification of Nilaparvata species is crucial for N. lugens forecasting and management. The traditional morphological identification method is not feasible for subadults and damaged specimens. Furthermore, this error-prone morphological identification method is time and labor intensive, with the need for expertise and experience. Here, we established a direct multiplex polymerase chain reaction (dmPCR) assay using crude tissue fluid as a template, omitting purified DNA extraction. The crude tissue fluid can be obtained by grinding specimens without any biological reagent but only using distilled water. This dmPCR assay, using three pairs of diagnostic primers, is based on internal transcribed spacers (ITS). Each primer pair amplifies a species-specific fragment of a different size, which were easily and reliably separated in a 2% agarose gel. Furthermore, the dmPCR was verified to be applicable to damaged tissue specimens, such as head, thorax, or abdomen. In conclusion, this dmPCR assay is a novel, time-saving, cost-effective, and easy-to-apply molecular diagnostic method for the identification of the above three sibling species, N. lugens, N. bakeri, and N. muiri.
褐飞虱（Nilaparvata lugens (Stål)，半翅目：飞虱科）是亚洲一种具有破坏性的水稻害虫。目前，褐飞虱的一种重要监测方法是通过黑光灯诱捕。然而，褐飞虱的两个近缘种，即拟褐飞虱（Nilaparvata bakeri (Muri)）和穆氏飞虱（Nilaparvata muiri China），也能被黑光灯诱捕，而这些物种仅以禾本科杂草为食，不以水稻为食。因此，准确鉴定褐飞虱属物种对于褐飞虱的预测和管理至关重要。传统的形态学鉴定方法对于若虫和受损标本不可行。此外，这种容易出错的形态学鉴定方法既耗时又费力，还需要专业知识和经验。在此，我们建立了一种直接多重聚合酶链反应（dmPCR）检测方法，以粗组织液为模板，省略了纯化DNA的提取步骤。粗组织液可以通过研磨标本获得，无需任何生物试剂，仅使用蒸馏水即可。这种基于内转录间隔区（ITS）的dmPCR检测方法使用三对诊断引物。每对引物扩增出大小不同的物种特异性片段，这些片段在2%琼脂糖凝胶中易于且可靠地分离。此外，已验证dmPCR适用于受损组织标本，如头部、胸部或腹部。总之，这种dmPCR检测方法是一种新颖、省时、经济高效且易于应用的分子诊断方法，用于鉴定上述三种近缘种，即褐飞虱、拟褐飞虱和穆氏飞虱。
