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

Singh, O. P., Bali, P., Hemingway, Janet ORCID:, Subbarao, S. K., Dash, A. P. and Adak, T. (2009) 'PCR-based methods for the detection of L1014 kdr mutation in Anopheles culicifacies sensu lato'. Malaria Journal, Vol 8, p. 8.

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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.

Item Type: Article
Subjects: WC Communicable Diseases > Tropical and Parasitic Diseases > WC 755 Epidemiology
QX Parasitology > Insects. Other Parasites > QX 515 Anopheles
WB Practice of Medicine > Medical Climatology > WB 710 Diseases of geographic areas
WA Public Health > Preventive Medicine > WA 110 Prevention and control of communicable diseases. Transmission of infectious diseases
WA Public Health > Preventive Medicine > WA 240 Disinfection. Disinfestation. Pesticides (including diseases caused by)
WC Communicable Diseases > Tropical and Parasitic Diseases > WC 765 Prevention and control
QX Parasitology > Insects. Other Parasites > QX 510 Mosquitoes
QW Microbiology and Immunology > QW 45 Microbial drug resistance. General or not elsewhere classified.
WA Public Health > WA 105 Epidemiology
Digital Object Identifer (DOI):
Depositing User: Users 183 not found.
Date Deposited: 10 Mar 2010 14:16
Last Modified: 24 Jan 2022 15:12


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