重大潜在入侵害虫番茄潜叶蛾的SS-COⅠ快速检测技术

Species-specific CO Ⅰ primers for rapid identification of Tuta absoluta (Meyrick), a significant, potential alien species

【背景】番茄潜叶蛾是源自南美洲的一种最具毁灭性的世界性入侵害虫,其适生区范围广,且与其他潜叶类昆虫难以准确、快速识别。【方法】以田间常见的其他6种潜叶类害虫为对照,采用基于mtDNA COⅠ基因的种特异性SS-COⅠ方法,研究番茄潜叶蛾快速分子检测技术。【结果】种特异性检测结果显示,SS-COⅠ引物只对番茄潜叶蛾mtDNA具有扩增能力,对美洲斑潜蝇、南美斑潜蝇、三叶草斑潜蝇、葱斑潜蝇、豌豆潜叶蝇、桃潜叶蛾等其他潜叶类害虫不具有扩增效果。该引物不仅对成虫和单粒卵具有很好的扩增效能,对单根触角、头部、胸部、腹部以及翅和足等成虫残体亦具有同样的扩增能力,其最低检出阈值为312.5pg·μL-1。【结论与意义】该方法在有效防范番茄潜叶蛾侵入我国,保障农业生产安全和生态环境安全中意义重大。

[ Background]The tomato leafminer Tuta absoluta （Meyrick）, native to South America, is one of the most devastating and invasive pest species worldwide. Morphological identification of T. absoluta is limited by small size, the high degree of similarity and polymorphism. [ Method] In this study, a method was described for the development of DNA marker for identification of T. abso- luta. One pair of species-specific PCR primers （SS-COI） based on a fragment of known mitochondrial DNA cytochrome oxidase I （mtDNA COI） sequence was designed for T. absoluta. The specificity of the primer pair was validated using six other leafminer species, including Liriomyza sativae Blanchard, L. huidobrensis （Blanchard）, L. trifolli （Brugess）, L. chinensis （Kato）, Phytomyza horticola Goureau and Lyonetia clerkella L. , all of them commonly occurring in China. [ Result ] All T. absoluta specimens were detected, and no cross reactions with other leafminer species were observed. The method was tested on single egg and adult debris （including antenna, head, thorax, abdomen, wing, leg） of T. absoluta, and proved to be applicable. Even at the concentration of 312.5 pg · μL^-1 DNA, the target fragment could be detected in all replicates. [ Conclusion and significance ] The technique developed here should be useful in quarantine at ports of entry and in guarantee the safety of agricultural production and ecological environment in China.