Zhang_1999_Pestic.Biochem.Physiol_65_25

An A to G point mutation that results in a serine to glycine amino acid change (S291G) in the acetylcholinesterase (AChE, EC 3.1.1.7) gene was identified previously as associated with azinphosmethyl resistance in Colorado potato beetle due to target site insensitivity. To efficiently validate the detection process of the S291G mutation and base the DNA diagnostic method on direct determination of nucleic acid sequence, a single-stranded conformational polymorphism (SSCP) protocol and a minisequencing reaction were developed. SSCP protocols using a 163-bp DNA template that spans the mutation resulted in an easy, rapid, cheap, and rugged DNA-based diagnostic method, which was capable of separating azinphosmethyl-susceptible and - resistant beetles. For minisequencing, PCR-amplified and biotinylated DNA templates from both susceptible and resistant beetles, which contain the mutation site, were bound to streptavidin-coated microplate strips. Minisequencing was accomplished with a detection primer that annealed adjacent to the point mutation, digoxigenin-labeled dATP, or alternatively, digoxigenin-labeled dUTP and AmpliTaq polymerase. The sequencing reaction added a digoxigenin-labeled dATP only when matched to the biotinylated DNA template (dATP and 3'...GGTCA...5'). Digoxigenin-labeled DNA was detected using peroxidase-conjugated digoxigenin antibodies and quantitated as optical density (OD) at 450 nm in a microplate reader. The OD readings obtained with digoxigenin-labeled dATP in the presence of susceptible AChE DNA template was 0.319 +/- 0.05, which was significantly higher than that obtained in the presence of the azinphosmethyl-resistant template (0.031 +/- 0.018) (P < 0.001). These highly significant results agree well with the susceptibility of AChE from individual insects as judged by AChE inhibition by azinphosmethyl-oxon and further support the contention that A to G point mutation, which occurs only in AChE gene of azinphosmethyl-resistant beetles, is responsible for enzyme insensitivity. Compared with SSCP, the minisequencing reaction provides a direct means to validate this specific point mutation. Coupling minisequencing with the ease and durability of SSCP will allow us to determine the presence or absence of the S291G mutation in an efficient and unambiguous manner. As such, similar approaches could be used to validate point mutations in any resistant strain of insect.