Jiao Jipeng, Ren Lili, Chen Rumin, Tao Jing, Luo Youqing
Key Laboratory for Forest Pest Control, College of Forestry, Beijing Forestry University, Beijing 100083, China.
Sino-French Joint Laboratory for Invasive Forest Pests in Eurasia, Beijing Forestry University-French National Research Institute for Agriculture, Food and Environment (INRAE), Beijing 100083, China.
Insects. 2022 Jun 11;13(6):540. doi: 10.3390/insects13060540.
Pine needle gall midge (), native to Japan, has become a serious invasive pest in South Korea and, more recently in 2006, in China. It was first discovered in Qingdao, Shandong Province, and has caused serious damage to local . The insect's small size makes morphological-based identification difficult; therefore, molecular detection techniques are urgently needed for monitoring and preventing its further spread. At present, there is no simple and accurate field molecular identification tool. To solve this problem, a LAMP-based molecular diagnosis technology of was developed. Four LAMP primers were designed to specifically amplify DNA. Positive LAMP reactions usually produce amplification in one hour. The optimal incubation conditions for LAMP detection were determined with 4 LAMP primers for 60 min at 61 °C. The LAMP detection range of gDNA concentrations is wide, with a minimum detectable gDNA concentration of 300 fg. A non-destructive DNA-releasing procedure, HotSHOT "HS6", which could extract "crude DNA" for LAMP assay in 10 min, was used for larval and adult samples. Therefore, we established a LAMP-based rapid molecular identification method that can be applied in the monitoring and management of .
松针瘿蚊原产于日本，已成为韩国的一种严重入侵害虫，最近在2006年，在中国也出现了。它首次在山东省青岛市被发现，并对当地造成了严重破坏。这种昆虫体型小，基于形态学的鉴定困难；因此，迫切需要分子检测技术来监测和防止其进一步传播。目前，还没有简单准确的现场分子鉴定工具。为了解决这个问题，开发了一种基于环介导等温扩增技术（LAMP）的松针瘿蚊分子诊断技术。设计了4条LAMP引物来特异性扩增松针瘿蚊的DNA。阳性LAMP反应通常在1小时内产生扩增。使用4条LAMP引物在61℃下孵育60分钟确定了LAMP检测的最佳条件。LAMP对基因组DNA（gDNA）浓度的检测范围很宽，最低可检测gDNA浓度为300飞克。一种非破坏性DNA释放程序HotSHOT“HS6”，可在10分钟内提取用于LAMP检测的“粗DNA”，用于幼虫和成虫样本。因此，我们建立了一种基于LAMP的快速分子鉴定方法，可应用于松针瘿蚊的监测和管理。
