Detection of knockdown resistance (kdr) mutations in Anopheles gambiae:: a comparison of two new high-throughput assays with existing methods

Background: Knockdown resistance ( kdr) is a well- characterized mechanism of resistance to pyrethroid insecticides in many insect species and is caused by point mutations of the pyrethroid target site the para- type sodium channel. The presence of kdr mutations in Anopheles gambiae, the most important malaria vector in Africa, has been monitored using a variety of molecular techniques. However, there are few reports comparing the performance of these different assays. In this study, two new high- throughput assays were developed and compared with four established techniques. Methods: Fluorescence- based assays based on 1) TaqMan probes and 2) high resolution melt ( HRM) analysis were developed to detect kdr alleles in An. gambiae. Four previously reported techniques for kdr detection, Allele Specific Polymerase Chain Reaction ( AS- PCR), Heated Oligonucleotide Ligation Assay ( HOLA), Sequence Specific Oligonucleotide Probe - Enzyme- Linked ImmunoSorbent Assay ( SSOP- ELISA) and PCR- Dot Blot were also optimized. The sensitivity and specificity of all six assays was then compared in a blind genotyping trial of 96 single insect samples that included a variety of kdr genotypes and African Anopheline species. The relative merits of each assay was assessed based on the performance in the genotyping trial, the length/ difficulty of each protocol, cost ( both capital outlay and consumable cost), and safety ( requirement for hazardous chemicals). Results: The real- time TaqMan assay was both the most sensitive ( with the lowest number of failed reactions) and the most specific ( with the lowest number of incorrect scores). Adapting the TaqMan assay to use a PCR machine and endpoint measurement with a fluorimeter showed a slight reduction in sensitivity and specificity. HRM initially gave promising results but was more sensitive to both DNA quality and quantity and consequently showed a higher rate of failure and incorrect scores. The sensitivity and specificity of AS- PCR, SSOP- ELISA, PCR Dot Blot and HOLA was fairly similar with a small number of failures and incorrect scores. Conclusion: The results of blind genotyping trials of each assay indicate that where maximum sensitivity and specificity are required the TaqMan real- time assay is the preferred method. However, the cost of this assay, particularly in terms of initial capital outlay, is higher than that of some of the other methods. TaqMan assays using a PCR machine and fluorimeter are nearly as sensitive as real- time assays and provide a cost saving in capital expenditure. If price is a primary factor in assay choice then the AS- PCR, SSOP- ELISA, and HOLA are all reasonable alternatives with the SSOP- ELISA approach having the highest throughput.