title: Detection of G119S ace-1 (R) mutation in field-collected Anopheles gambiae mosquitoes using allele-specific loop-mediated isothermal amplification (AS-LAMP) method.
abstract: BACKGROUND
  Malaria vectors have developed resistance to the four families of insecticides available for public health purposes. For example, the kdr mutation is associated with organochlorines and pyrethroids resistance. It is of particular concern that organophosphate and carbamate resistance associated with the G119S ace-1 (R) mutation has recently increased in West Africa in extent and frequency, and is now spreading through the Anopheles gambiae malaria vector population. There is an urgent need to improve resistance management using existing insecticides and new tools to quickly assess resistance level for rapid decision-making.
  
  
  METHODS
  DNA extracted from field-collected mosquitoes was used to develop the method. Specific primers were designed manually to match the mutation region and an additional mismatched nucleotide in the penultimate position to increase specificity. Other primers used are common to both wild and mutant types. The allele specific (AS)-LAMP method was compared to the PCR restriction fragment length polymorphism (PCR-RFLP) and real-time PCR (RT-PCR) methods using the genomic DNA of 104 field-collected mosquitoes.
  
  
  RESULTS
  The primers designed for LAMP were able to distinguish between the wild type (ace-1 (S) ) and mutated type allele (ace-1 (R) ). Detection time was 50 min for the wild type homozygous and 64 min for the heterozygous. No amplification of the resistant allele took place within the 75-min test period when using the wild type primers. For the ace-1 (R) resistant type, detection time was 51 min for the resistant homozygous and 55 min for the heterozygous. No amplification of the wild type allele took place within the 75-min test period when using the resistant type primers. Gel electrophoresis of LAMP products confirmed that amplification was primer-DNA specific, i.e., primers could only amplify their target specific DNA. AS-LAMP, PCR-RFLP, and RT-PCR showed no significant difference in the sensitivity and specificity of their ace-1 (R) detection ability.
  
  
  CONCLUSIONS
  The AS-LAMP method could detect the ace-1 (R) mutation within 60 min, which is faster than conventional PCR-RFLP. This method may be used to quickly detect the ace-1 (R) mutation for rapid decision-making, even in less well-equipped laboratories.
authors.affiliation: National Research Centre for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, 080-8555, Japan. a.badolo@gmail.com.|Centre National de Recherche et de Formation sur le Paludisme (CNRFP), BP 2208, Ouagadougou 01, Burkina Faso. a.badolo@gmail.com.|Laboratoire d'Entomologie Fondamentale et Appliquée, Université de Ouagadougou, BP 7021, Ouagadougou 03, Burkina Faso. a.badolo@gmail.com.
authors.forename: Athanase
authors.initials: A
authors.lastname: Badolo
authors.affiliation: National Research Centre for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, 080-8555, Japan. s15200@st.obihiro.ac.jp.
authors.forename: Hironori
authors.initials: H
authors.lastname: Bando
authors.affiliation: Centre National de Recherche et de Formation sur le Paludisme (CNRFP), BP 2208, Ouagadougou 01, Burkina Faso. traorealphonse@yahoo.fr.
authors.forename: Alphonse
authors.initials: A
authors.lastname: Traoré
authors.affiliation: National Research Centre for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, 080-8555, Japan. m-kouketsu@obihiro.ac.jp.
authors.forename: Mami
authors.initials: M
authors.lastname: Ko-Ketsu
authors.affiliation: Centre National de Recherche et de Formation sur le Paludisme (CNRFP), BP 2208, Ouagadougou 01, Burkina Faso. guelbeogo.cnrfp@fasonet.bf.
authors.forename: Wamdaogo Moussa
authors.initials: WM
authors.lastname: Guelbeogo
authors.affiliation: Department of Tropical Medicine, The Jikei University School of Medicine, Tokyo, 105-8461, Japan. kanuka@jikei.ac.jp.
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Kanuka
authors.affiliation: Vector Group, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA, UK. Hilary.Ranson@lstmed.ac.uk.
authors.forename: Hilary
authors.initials: H
authors.lastname: Ranson
authors.affiliation: Centre National de Recherche et de Formation sur le Paludisme (CNRFP), BP 2208, Ouagadougou 01, Burkina Faso. n.fale.cnlp@fasonet.bf.
authors.forename: N'Falé
authors.initials: N
authors.lastname: Sagnon
authors.affiliation: National Research Centre for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, 080-8555, Japan. fukumoto@obihiro.ac.jp.
authors.forename: Shinya
authors.initials: S
authors.lastname: Fukumoto
mesh_terms: D000110:Acetylcholinesterase / Q000235:genetics*; D000354:Africa, Western; D000818:Animals; D000852:Anopheles / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004772:Entomology / Q000379:methods*; D005260:Female; D006801:Humans; D007303:Insect Vectors / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D007305:Insecticide Resistance; D050505:Mutant Proteins / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D013997:Time Factors
chemical_list: D017931:DNA Primers; D050505:Mutant Proteins; D000110:Acetylcholinesterase