入侵害虫麦秆蝇(Elachiptera decipiens)LAMP检测方法的建立OA

Elachiptera decipiens,an invasive insect pest recently identified in Xinjiang,damages wheat stems with its larvae and causes symptoms similar to those of other chloropid pests,making accurate differentiation difficult and complicating population monitoring.This study aimed to develop a rapid and visual detection method based on loop-mediated isothermal amplification(LAMP).The specific LAMP primers targeting the mitochondrial cytochrome oxidase Ⅰ gene(COⅠ)of E.decipiens were designed.The LAMP assay was then optimized,and the specificity and sensitivity of the primers were evaluated.A DNA toothpick extraction method combined with the optimized LAMP system was em-ployed for visual identification of samples.The results showed that the LAMP primers exhibited high specificity,and the optimized LAMP reaction system was as follows:2 μL of 10×Acculso Bca Buffer,1 μL of MgSO4(100 mmol/L),3.5 μL of dNTP Mix,1 μL of FIP/BIP(40 μmol/L),0.5 μL of F3/B3(10 μmol/L),3 μL of betaine(5 mol/L),1 μL of Bca DNA Polymerase,1 μL of Hydroxynaphthol Blue(250 μmol/L),9.5 μL of enzyme-free sterile water,and 1 μL of DNA template.The LAMP assay amplified positive samples within 50 min at constant-temperature 65℃,with a sensitive of very low levels of DNA(1×10-3 ng/μL).Combined with DNA toothpick extraction,the detection for samples could be completion within 1 h.Field sample testing demonstrates that the visual LAMP protocol is efficient,sensitive and simple to operate,satisfying the requirements for on-site detection during pest surveillance.