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Background   Anopheles culicifacies s.l ., a major malaria vector in India, has developed widespread resistance to DDT and is becoming resistant to pyrethroids–the only insecticide class recommended for the impregnation of bed nets. Knock-down resistance due to a point mutation in the voltage gated sodium channel at L1014 residue ( kdr ) is a common mechanism of resistance to DDT and pyrethroids. The selection of this resistance may pose a serious threat to the success of the pyrethroid-impregnated bed net programme. This study reports the presence of  kdr  mutation (L1014F) in a field population of  An. culicifacies s.l . and three new PCR-based methods for  kdr  genotyping.    Methods  The IIS4-IIS5 linker to IIS6 segments of the para type voltage gated sodium channel gene of DDT and pyrethroid resistant  An. culicifacies s.l . population from the Surat district of India was sequenced. This revealed the presence of an A-to-T substitution at position 1014 leading to a leucine-phenylalanine mutation (L1014F) in a few individuals. Three molecular methods viz. Allele Specific PCR (AS-PCR), an Amplification Refractory Mutation System (ARMS) and Primer Introduced Restriction Analysis-PCR (PIRA-PCR) were developed and tested for  kdr  genotyping. The specificity of the three assays was validated following DNA sequencing of the samples genotyped.    Results  The genotyping of this  An. culicifacies s.l . population by the three PCR based assays provided consistent result and were in agreement with DNA sequencing result. A low frequency of the  kdr  allele mostly in heterozygous condition was observed in the resistant population. Frequencies of the different genotypes were in Hardy-Weinberg equilibrium.    Conclusion  The Leu-Phe mutation, which generates the  kdr  phenotype in many insects, was detected in a pyrethroid and DDT resistant  An. culicifacies s.l . population. Three PCR-based methods were developed for  kdr  genotyping. All the three assays were specific. The ARMS method was refractory to non-specific amplification in non-stringent amplification conditions. The PIRA-PCR assay is able to detect both the codons for the phenylalanine mutation at  kdr  locus, i.e., TTT and TTC, in a single assay, although the latter codon was not found in the population genotyped.

PCR-based methods for the detection of L1014 kdr mutation in Anopheles culicifacies sensu lato