An efficient strategy for validation of a point mutation associated with acetylcholinesterase sensitivity to azinphosmethyl in Colorado potato beetle

An A to G point mutation that results in a serine to glycine amino acid cha nge (S291G) in the acetylcholinesterase (AChE, EC 3.1.1.7) gene was identif ied previously as associated with azinphosmethyl resistance in Colorado pot ato beetle due to target site insensitivity. To efficiently validate the de tection process of the S291G mutation and base the DNA diagnostic method on direct determination of nucleic acid sequence, a single-stranded conformat ional polymorphism (SSCP) protocol and a minisequencing reaction were devel oped. SSCP protocols using a 163-bp DNA template that spans the mutation re sulted in an easy, rapid, cheap, and rugged DNA-based diagnostic method, wh ich was capable of separating azinphosmethyl-susceptible and - resistant be etles. Far minisequencing, PCR-amplified and biotinylated DNA templates fro m both susceptible and resistant beetles, which contain the mutation site, were bound to streptavidin-coated microplate strips. Minisequencing was acc omplished with a detection primer that annealed adjacent to the point mutat ion, digoxigenin-labeled dATP, or alternatively, digoxigenin-labeled dUTP a nd AmpliTaq polymerase. The sequencing reaction added a digoxigenin-labeled dATP only when matched to the biotinylated DNA template (dATP and 3'...GGT CA...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 dAT P in the presence of susceptible AChE DNA template was 0.319 +/- 0.05, whic h was significantly higher than that obtained in the presence of the azinph osmethyl-resistant template (0.031 +/- 0.018) (P < 0.001). These highly sig nificant results agree well with the susceptibility of AChE from individual insects as judged by AChE inhibition by azinphosmethyl-oxon and further su pport the contention that A to G point mutation, which occurs only in AChE gene of azinphosmethyl-resistant beetles, is responsible for enzyme insensi tivity. Compared with SSCP, the minisequencing reaction provides a direct m eans 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 s uch, similar approaches could be used to validate point mutations in any re sistant strain of insect. (C) 1999 Academic Press.