%0 Journal Article
%T PCR-based methods for the detection of L1014 kdr mutation in Anopheles culicifacies sensu lato
%A Om P Singh
%A Prerna Bali
%A Janet Hemingway
%A Sarala K Subbarao
%A Aditya P Dash
%A Tridibes Adak
%J Malaria Journal
%D 2009
%I BioMed Central
%R 10.1186/1475-2875-8-154
%X 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.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.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.Anopheles culicifacies s.l. is the main malaria vector in the Indian subcontinent, affecting mainly rural areas, and contributes 60¨C65% of malaria cases in India [1]. As this is an endophilic vector, indoor residual spraying (IRS) of DDT is the main strategy used for its control. This species is resistant to DDT in most parts of India. Although DDT is banned in many countries, the recent endorsement by World Health Organization for
%U http://www.malariajournal.com/content/8/1/154