Nam Hwa Yeun, Kim Ju Hyeon, Lee Si Hyeock, Heckel David G, Kim Juil
Agriculture and Life Science Research Institute, Kangwon National University, Chuncheon 24341, Korea.
Research Institute of Agriculture and Life Science, Seoul National University, Seoul 08826, Korea.
Insects. 2021 Sep 29;12(10):883. doi: 10.3390/insects12100883.
Molecular-based species identification tools are helpful to identify tiny insect and lepidopteran pests that show morphological similarities in the larval stage and are essential for quarantine as well as agricultural research. Here, we focused on four major pests: , , , and . and mitochondrial genome sequences were newly identified and species-specific sequence regions were identified in the cytochrome c oxidase subunit II and III regions. Species primers were designed and applied in loop-mediated isothermal amplification (LAMP) and PCR to identify Korean field-collected or overseas samples. The optimal incubation conditions for LAMP were 61 °C for 60 min with four LAMP primers. Additional loop primers increased the amplification efficiency for , and the nonspecific amplification for other species. The LAMP assay could detect a wide range of DNA concentrations, with the range 1 ng-1 pg in dependence of four LAMP primers. The DNA-releasing technique, without DNA extraction, in the LAMP assay involved larval or adult tissue sample incubation at 95 °C for 5 min. The entire process takes approximately 70 min. This new molecular diagnostic method is simple and accurate, with application in the field and laboratory and for monitoring and ecological studies.
基于分子的物种鉴定工具有助于识别在幼虫阶段表现出形态相似性的微小昆虫和鳞翅目害虫，这对于检疫以及农业研究至关重要。在此，我们聚焦于四种主要害虫： 、 、 和 。新鉴定了 和 的线粒体基因组序列，并在细胞色素c氧化酶亚基II和III区域鉴定了物种特异性序列区域。设计了物种引物并将其应用于环介导等温扩增（LAMP）和PCR中，以鉴定韩国野外采集的或海外的样本。LAMP的最佳孵育条件是使用四种LAMP引物在61°C下孵育60分钟。额外的环引物提高了 的扩增效率，并增加了其他物种的非特异性扩增。LAMP检测可以检测广泛的DNA浓度范围，取决于四种LAMP引物，范围为1 ng - 1 pg。LAMP检测中无需DNA提取的DNA释放技术涉及将幼虫或成虫组织样本在95°C下孵育5分钟。整个过程大约需要70分钟。这种新的分子诊断方法简单准确，可应用于野外和实验室以及监测和生态学研究。
