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

Lynd, Amy ORCID: https://orcid.org/0000-0001-6054-0525, Ranson, Hilary ORCID: https://orcid.org/0000-0003-2332-8247, McCall, Philip ORCID: https://orcid.org/0000-0002-0007-3985, Randle, Nadine P., Black, W. C., Walker, E. D. and Donnelly, Martin ORCID: https://orcid.org/0000-0001-5218-1497 (2005) 'A simplified high-throughput method for pyrethroid knock-down resistance (kdr) detection in Anopheles gambiae'. Malaria Journal, Vol 4, p. 16.

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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.

Item Type:	Article
Additional Information:	The electronic version of this article is the complete one and can be found online at: http://www.malariajournal.com/content/4/1/16
Uncontrolled Keywords:	permethrin-impregnated bednets polymerase chain-reaction sodium-channel gene identification populations mortality malaria differentiation sequence mutation
Subjects:	QX Parasitology > Insects. Other Parasites > QX 515 Anopheles QX Parasitology > Insects. Other Parasites > QX 600 Insect control. Tick control
Faculty: Department:	Groups (2002 - 2012) > Vector Group
Digital Object Identifer (DOI):	https://doi.org/10.1186/1475-2875-4-16
Depositing User:	Ms Julia Martin
Date Deposited:	25 Oct 2011 10:50
Last Modified:	16 Sep 2019 09:16
URI:	https://archive.lstmed.ac.uk/id/eprint/1939

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