A comparison of DNA sequencing and the hydrolysis probe analysis (TaqMan assay) for knockdown resistance ( kdr ) mutations in Anopheles gambiae from the Republic of the Congo

• Published in: Malaria Journal
• Main Authors: Coetzee Maureen, Spillings Belinda L, Choi Kwang, Hunt Richard H, Koekemoer Lizette L
• Format: Article in Journal/Newspaper
• Language: English
• Published: BMC 2010
• Subjects: Arctic medicine. Tropical medicine; RC955-962; Infectious and parasitic diseases; RC109-216; Arctic; Noire; Pointe Noire
• Online Access: https://doi.org/10.1186/1475-2875-9-278; https://doaj.org/article/7b9609f5e50a410e9cfb82c886a28573

Abstract Background Knockdown resistance ( kdr ) caused by a single base pair mutation in the sodium channel gene is strongly associated with pyrethroid insecticide resistance in Anopheles gambiae in West-Central Africa. Recently, various molecular techniques have been developed to screen for the presence of the kdr mutations in vector populations with varying levels of accuracy. In this study, the results of the hydrolysis probe analysis for detecting the kdr mutations in An. gambiae s.s. from the Republic of the Congo were compared with DNA sequence analysis. Methods A total of 52 pyrethroid and DDT resistant An. gambiae from Pointe-Noire (Congo-Brazzaville) were tested for detection of the two kdr mutations ( kdr -e and kdr -w) that are known to occur in this species. Results from the hydrolysis probe analysis were compared to DNA sequencing to verify the accuracy of the probe analysis for this vector population. Results Fifty-one specimens were found to be An. gambiae S-form and one was a M/S hybrid. DNA sequencing revealed that more than half of the specimens (55.8%) carried both the kdr -e and kdr -w resistance mutations, seven specimens (13.5%) were homozygous for the kdr -e mutation, and 14 specimens (26.9%) were homozygous for the kdr -w mutation. A single individual was genotyped as heterozygous kdr -e mutation (1.9%) only and another as heterozygous kdr -w mutation (1.9%) only. Analysis using hydrolysis probe analysis, without adjustment of the allelic discrimination axes on the scatter plots, revealed six specimens (11.5%) carrying both mutations, 30 specimens (57.8%) as homozygous kdr -w, six specimens (11.5%) homozygous for the kdr -e mutation, one specimen (1.9%) heterozygous for the kdr -w mutation and one specimen (1.9%) present in wild type form. Eight of the specimens (15.4%) could not be identified using unadjusted hydrolysis probe analysis values. No heterozygous kdr -e mutations were scored when adjustment for the allelic discrimination axes was omitted. However, when the axes on the ...