A simplified high-throughput method for pyrethroid knock-down resistance (kdr) detection in Anopheles gambiae

Amy Lynd1, Hilary Ranson1, Philip J. McCall1, Nadine Randle1, William C. Black2, Edward D. Walker3, Martin J. Donnelly1
1Vector Research Group, Liverpool Tropical School of Medicine, Pembroke Place, Liverpool, UK
2Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USA
3Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA

Tóm tắt

Abstract Background A single base pair mutation in the sodium channel confers knock-down resistance to pyrethroids in many insect species. Its occurrence in Anopheles mosquitoes may have important implications for malaria vector control especially considering the current trend for large scale pyrethroid-treated bednet programmes. Screening Anopheles gambiae populations for the kdr mutation has become one of the mainstays of programmes that monitor the development of insecticide resistance. The screening is commonly performed using a multiplex Polymerase Chain Reaction (PCR) which, since it is reliant on a single nucleotide polymorphism, can be unreliable. Here we present a reliable and potentially high throughput method for screening An. gambiae for the kdr mutation. Methods A Hot Ligation Oligonucleotide Assay (HOLA) was developed to detect both the East and West African kdr alleles in the homozygous and heterozygous states, and was optimized for use in low-tech developing world laboratories. Results from the HOLA were compared to results from the multiplex PCR for field and laboratory mosquito specimens to provide verification of the robustness and sensitivity of the technique. Results and Discussion The HOLA assay, developed for detection of the kdr mutation, gives a bright blue colouration for a positive result whilst negative reactions remain colourless. The results are apparent within a few minutes of adding the final substrate and can be scored by eye. Heterozygotes are scored when a sample gives a positive reaction to the susceptible probe and the kdr probe. The technique uses only basic laboratory equipment and skills and can be carried out by anyone familiar with the Enzyme-linked immunosorbent assay (ELISA) technique. A comparison to the multiplex PCR method showed that the HOLA assay was more reliable, and scoring of the plates was less ambiguous. Conclusion The method is capable of detecting both the East and West African kdr alleles in the homozygous and heterozygous states from fresh or dried material using several DNA extraction methods. It is more reliable than the traditional PCR method and may be more sensitive for the detection of heterozygotes. It is inexpensive, simple and relatively safe making it suitable for use in resource-poor countries.

Từ khóa


Tài liệu tham khảo

Phillips-Howard PA, Nahlen BL, Kolczak MS, Hightower AW, ter Kuile FO, Alaii JA, Gimnig JE, Arudo J, Vulule JM, Odhacha A, Kachur SP, Schoute E, Rosen DH, Sexton JD, Oloo AJ, Hawley WA: Efficacy of permethrin-treated bed nets in the prevention of mortality in young children in an area of high perennial malaria transmission in western Kenya. Am J Trop Med Hyg. 2003, 68 (4): 23-29.

Binka FN, Kubaje A, Adjuik M, Williams LA, Lengeler C, Maude GH, Armah GE, Kajihara B, Adiamah JH, Smith PG: Impact of permethrin impregnated bednets on child mortality in Kassena-Nankana district, Ghana: A randomized controlled trial. Tropical Medicine and International Health. 1996, 1 (2): 147-154.

D' Alessandro U, Olaleye BO, McGuire W, Langerock P, Bennett S, Aikins MK, Thomson MC, Cham MK, Cham BA, Greenwood BM: Mortality and morbidity from malaria in Gambian children after introduction of an impregnated bednet program. Lancet. 1995, 345 (8948): 479-483. 10.1016/S0140-6736(95)90582-0.

Hemingway J, Ranson H: Insecticide resistance in insect vectors of human disease. Annu Rev Entomol. 2000, 45: 371-391. 10.1146/annurev.ento.45.1.371.

Elissa N, Mouchet J, Riviere F, Meunier JY, Yao K: Resistance of Anopheles gambiae to pyrethroids in Cote d'Ivoire. Annales de la Societe Belge de Medecine Tropicale. 1993, 73: 291-294.

Vulule JM, Beach RF, Ayieli FK, Roberts JM, Mount DL, Mwangi RW: Reduced susceptibilty of Anopheles gambiae to permethrin associated with the use of permethrin-impregnated bednets and curtains in Kenya. Med Vet Entomol. 1994, 8: 71-75.

Mouchet J: Agriculture and vector resistance. Insect Sci Applic. 1988, 9 (3): 297-302.

Martinez-Torres D, Chandre F, Williamson MS, Darriet F, Berge JB, Devonshire AL, Guillet P, Pasteur N, Pauron D: Molecular characterization of pyrethroid knockdown resistance (kdr) in the major malaria vector Anopheles gambiae s.s. Insect Mol Biol. 1998, 7 (2): 179-184. 10.1046/j.1365-2583.1998.72062.x.

Chandre F, Darriet F, Duchon S, Finot L, Manguin S, Carnevale P, Guillet P: Modifications of pyrethroid effects associated with kdr mutation in Anopheles gambiae. Med Vet Entomol. 2000, 14 (1): 81-88. 10.1046/j.1365-2915.2000.00212.x.

Chandre F, Darrier F, Manga L, Akogbeto M, Faye O, Mouchet J, Guillet P: Status of pyrethroid resistance in Anopheles gambie sensu lato. Bulletin of the World Health Organization. 1999, 77 (3): 230-234.

Gentile G, Santolamazza F, Fanello C, Petrarca V, Caccone A, della Torre A: Variation in an intron sequence of the voltage-gated sodium channel gene correlates with genetic differentiation between Anopheles gambiae s.s. molecular forms. Insect Mol Biol. 2004, 13 (4): 371-377. 10.1111/j.0962-1075.2004.00494.x.

Ranson H, Jensen B, Vulule JM, Wang X, Hemingway J, Collins FH: Identification of a point mutation in the voltage-gated sodium channel gene of Kenyan Anopheles gambiae associated with resistance to DDT and pyrethroids. Insect Mol Biol. 2000, 9 (5): 491-497. 10.1046/j.1365-2583.2000.00209.x.

Conway DJ, Roper C, Oduola AMJ, Arnot DE, Kremsner PG, Grobusch MP, Curtis CF, Greenwood BM: High recombination rate in natural populations of Plasmodium falciparum. Proceedings of the National Academy of Science. 1999, 96 (8): 4506-4511. 10.1073/pnas.96.8.4506.

Livak KJ: Organization and mapping of a sequence on the Drosophila melanogaster X and Y chromosomes that is transcribed during spermatogenesis. Genetics. 1984, 107 (4): 611-634.

Ballinger-Crabtree ME, Black IV WC, Miller BP: Use of genetic polymorphisms detected by the Random-Amplified Polymorphic DNA Polymerase Chain Reaction (RAPD PCR) for differentiation and identification of Aedes aegypti subspecies and populations. Am J Trop Med Hyg. 1992, 47 (6): 893-901.

Scott JA, Brogdon WG, Collins FH: Identification of single specimens of the Anopheles gambiae complex by the Polymerase Chain Reaction. Am J Trop Med Hyg. 1993, 49 (4): 520-529.

Stump AD, Atieli FK, Vulule JM, Besansky NJ: Dynamics of the pyrethroid knockdown resistance allele in western Kenyan populations of Anopheles gambiae in response to insecticide-treated bed net trials. Am J Trop Med Hyg. 2004, 70 (6): 591-596.