Detection of Mutation by Allele-Specific Loop-Mediated Isothermal Amplification (AS-LAMP)

  • Hiroka Aonuma
  • Athanase Badolo
  • Kiyoshi Okado
  • Hirotaka Kanuka
Part of the Methods in Molecular Biology book series (MIMB, volume 1039)


For effective control of pathogen-transmitting mosquitoes, precise surveillance data of mosquito distribution are essential. Recently, an increase of insecticide resistance due to the kdr mutation in Anopheles gambiae, a mosquito that transmits the malaria parasite, has been reported. With the aim of developing a simple and effective method for surveying resistant mosquitoes, LAMP was applied to the allele-specific detection of the kdr gene in An. gambiae. Allele-specific LAMP (AS-LAMP) method successfully distinguished the kdr homozygote from the heterozygote and the wild type. The robustness of AS-LAMP suggests its usefulness for routine identification of insects, not only mosquitoes but also other vectors and agricultural pests. Here we describe the method of AS-LAMP to detect mutation in Anopheles mosquitoes.

Key words

LAMP Mosquito Allele-specific kdr 



This study was supported in part by Grants-in-Aid for Scientific Research from the Japanese Ministry of Education, Science, Sports, Culture and Technology to H.K., and the Funding Program for the Next Generation of World-Leading Researchers (NEXT Program) to H.K. H.A. is a research fellow of the Japan Society for the Promotion of Science.


  1. 1.
    World Health Organization (2011) Vector control. World malaria report, Chapter 4. World Health Organization, GenevaGoogle Scholar
  2. 2.
    World Health Organization (2012) Launch of the global plan for insecticide resistance management in malaria vectors. Event report. World Health Organization, GenevaGoogle Scholar
  3. 3.
    Martinez-Torres D, Chandre F, Williamson MS, Darriet F, Bergé JB, Devonshire AL, Guillet P, Pasteur N, Pauron D (1998) Molecular characterization of pyrethroid knockdown resistance (kdr) in the major malaria vector Anopheles gambiae s.s. Insect Mol Biol 7:179–184PubMedCrossRefGoogle Scholar
  4. 4.
    Ranson H, Jensen B, Vulule JM, Wang X, Hemingway J, Collins FH (2000) 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 9:491–497PubMedCrossRefGoogle Scholar
  5. 5.
    Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28:E63PubMedCrossRefGoogle Scholar
  6. 6.
    Aonuma H, Suzuki M, Iseki H, Perera N, Nelson B, Igarashi I, Yagi T, Kanuka H, Fukumoto S (2008) Rapid identification of Plasmodium-carrying mosquitoes using loop-mediated isothermal amplification. Biochem Biophys Res Commun 376:671–676PubMedCrossRefGoogle Scholar
  7. 7.
    Aonuma H, Yoshimura A, Perera N, Shinzawa N, Bando H, Oshiro S, Nelson B, Fukumoto S, Kanuka H (2009) Loop-mediated isothermal amplification applied to filarial parasites detection in the mosquito vectors: Dirofilaria immitis as a study model. Parasit Vectors 2:15PubMedCrossRefGoogle Scholar
  8. 8.
    Perera N, Aonuma H, Yoshimura A, Teramoto T, Iseki H, Nelson B, Igarashi I, Yagi T, Fukumoto S, Kanuka H (2009) Rapid identification of virus-carrying mosquitoes using reverse transcription-loop-mediated isothermal amplification. J Virol Methods 156:32–36PubMedCrossRefGoogle Scholar
  9. 9.
    Iwasaki M, Yonekawa T, Otsuka K, Suzuki W, Nagamine K, Hase T, Tatsumi K, Horigome T, Notomi T, Kanda H (2003) Validation of the loop-mediated isothermal amplification method for single nucleotide polymorphism genotyping with whole blood. Genome Lett 2:119–126CrossRefGoogle Scholar
  10. 10.
    Fukuta S, Mizukami Y, Ishida A, Kanbe M (2006) Development of loop-mediated isothermal amplification (LAMP)-based SNP markers for shelf-life in melon (Cucumis melo L.). J Appl Genet 47:303–308PubMedCrossRefGoogle Scholar
  11. 11.
    Ikeda S, Takabe K, Inagaki M, Funakoshi N, Suzuki K (2007) Detection of gene point mutation in paraffin sections using in situ loop-mediated isothermal amplification. Pathol Int 57:594–599PubMedCrossRefGoogle Scholar
  12. 12.
    Badolo A, Okado K, Guelbeogo WM, Aonuma H, Bando H, Fukumoto S, Sagnon N, Kanuka H (2012) Development of an allele-specific, loop-mediated, isothermal amplification method (AS-LAMP) to detect the L1014F kdr-w mutation in Anopheles gambiae s. l. Malar J 11:227PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2013

Authors and Affiliations

  • Hiroka Aonuma
    • 1
  • Athanase Badolo
    • 2
    • 3
  • Kiyoshi Okado
    • 1
  • Hirotaka Kanuka
    • 1
  1. 1.Department of Tropical MedicineThe Jikei University School of MedicineTokyoJapan
  2. 2.University of OuagadougouOuagadougouBurkina Faso
  3. 3.Centre National de Recherche et de Formation sur le PaludismeOuagadougouBurkina Faso

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