Primer design for the voltage-gated sodium channel, the pyrethroids' target site, in the triatomine vector Triatoma infestans (Hemiptera: Reduviidae).

Abstract

Resistance to pyrethroid insecticides has become more frequent in triatomines, associated with point mutations in their target site, the voltage-gated sodium channel (VGSC). These mutations have been reported in Argentina, Bolivia and Mexico, by means of nested PCR. In Chile, the repeated intradomiciliary presence of the vector Triatoma infestans after spraying may be related to resistance; however, target-site mutations have yet to be evaluated. Using a partial sequence of the VGSC as a search query for the T. infestans genome, the complete VGSC gene sequence was obtained. Ten primer pairs were generated with Primer-BLAST and tested in silico by BLAST against the T. infestans genome and against the NCBI nucleotide database (nr) to discard those with low specificity. Conventional PCRs were performed with T. infestans' DNA and the remaining primers, selecting one pair based on sensitivity and the absence of nonspecific bands. The 993 bp sequenced product allows for the evaluation of the three point mutations reported in the VGSC of triatomines. Primer validation was performed with L925I mutation-positive samples from Argentina. Chilean samples from five localities were also amplified and Sanger sequenced in search of mutations. The L925I mutation was detected only in the Argentinean controls. None of the variants evaluated were found in the Chilean samples. These primers will allow rapid evaluation of point mutations in the VGSC, which may be associated with reduced affinity of pyrethroids for their target site; thereby facilitating insecticide resistance surveillance.