A LAMP Assay for the Detection of Thecodiplosis japonensis, an Alien Gall Midge Species Pest of Pine Trees

SIMPLE SUMMARY: Thecodiplosis japonensis is considered the most harmful pest to pines in South Korea. T. japonensis is a native species of Japan. Recently, T. japonensis was discovered in China and has caused serious damage to local pine trees. Due to the small size and little morphological difference with its related species, it is difficult to accurately identify T. japonensis by morphological methods. Accurate and efficient molecular identification methods are urgently needed to detect this invasive gall midge pest, yet there was no molecular identification method for T. japonensis. In this study, we developed a LAMP assay to detect T. japonensis based on the COI gene sequence. The LAMP assay could detect as little as 300 fg of gDNA. Using colorimetric amplification and a crude gDNA extraction method, the total procedure could be processed in 75 min. The method established in the study can be easily used in both laboratory and field conditions, enabling rapid molecular identification of T. japonensis. ABSTRACT: Pine needle gall midge (T. japonensis), 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 Pinus thunbergii. 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 T. japonensis was developed. Four LAMP primers were designed to specifically amplify T. japonensis 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 T. japonensis.