Loop-mediated isothermal amplification for the detection of R81T mutation in nAChR with crude genomic DNA extracted from individual Myzus persicae

Abstract

Myzus persicae is an important insect pest worldwide and is mainly controlled by chemical insecticides. The escalating use of neonicotinoid insecticides has resulted in resistance development. Arginine-to-threonine substitution (R81T) in the D loop region of the nicotinic acetylcholine receptor (nAChR) subunit β1 has been associated with imidacloprid resistance in M. persicae. A simple and fast method to detect the frequency and distribution of this mutation in field populations of M. persicae is important for effective pest management. A modified buffer was used to lyse the individual M. persicae to obtain crude genomic DNA as a template for LAMP reactions. Four sets of primers were designed to target a 641-bp fragment of nAChR in M. persicae. With the crude gDNA and special primers, the concentrations of reaction components, time and temperature were optimized. Therefore, the LAMP for the detection of the R81T mutation was processed at 60 °C for 45 min followed by 80 °C for 10 min to stop the reaction using a common hot bath. Four field-collected populations of M. persicae were used to confirm the application of LAMP for detecting the R81T mutation (both RR and RS genotypes) based on HNB visualization and gel electrophoresis. A rapid, visual and efficient LAMP was successfully developed to distinguish between wild and mutated individuals with crude gDNA as a template extracted from individual M. persicae. This provides an easy and quick method for detecting R81T mutations in field populations of M. persicae for resistance management.