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

Abstract

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.

Figure 1

Examples of AS-PCR products for kdr-e and kdr-w genotyping. Gels A to D show examples of AS-PCR results using four different protocols, A [29], B [11], C [12], D [20]. Gels E to G show the result of using different DNA polymerases on the AS-PCR method described by [20], E: Promega PCR master mix, F: Qiagen HotStar Taq G:Finzymes Dynazyme II Gel H is an example of results of using the protocol of [20] with the Promega PCR master mix to genotype samples using the kdr-e assay from the 96 reference plate and gel I for the kdr-w assay. The same DNA templates were used in PCRs shown in gels A to G and from left to right were 100 bp DNA Ladder (Fermentas), homozygous wildtype, homozygous wildtype, heterozygous, heterozygous, homozygous mutant, homozygous mutant.

Figure 2

Real-time TaqMan detection of the kdr-e and kdr-w alleles. A) and B) Detection of the kdr-w mutation. C) and D) Detection of the kdr-e mutation. A) Cycling of FAM-labelled probe specific for the kdr-w allele. C) Cycling of the FAM-labelled probe specific for the kdr-e allele. B) and D) cycling of the VIC labelled probe specific for the wild type allele. S: Wild type allele (L1014), Rw: Resistant allele, West African mutation (L1014F), Re: Resistant allele, East African mutation (L1014S).

Figure 3

Scatter plot analysis of TaqMan fluorescence data. In this example real time PCR was carried out using the east kdr assay on ~70 samples from the 96 samples reference plate then fluorescence values of the FAM labelled probe specific for the kdr-e mutation were plotted against the VIC labelled probe specific for the wild type allele.

Figure 4

Scatter plot analysis of TaqMan End point assay using a PCR machine+fluorimeter. In this example PCR was carried out using the west kdr assay from 48 samples of the 96 sample reference plate and the fluorescence of VIC and FAM was measured on a fluorimeter. The data was corrected for background and then plotted in a bi-directional scatter plot in Microsoft Excel. Values of X and Y axes are raw fluorescence values.

Figure 5

High Resolution Melt (HRM) for detection of kdr-e and kdr-w mutations. A) HRM detection of kdr-e allele. B) HRM detection of kdr-w allele. C) HRM detection for both kdr-e and kdr-w mutations. D) The melt curve profiles shown in C plotted as a difference plot as an aid to visual interpretation. In this case the difference in fluorescence of a sample to a selected control (in this case an S/Re genotype control) is plotted at each temperature transition. S: Wild type allele (L1014), Rw: Resistant allele, West African mutation (L1014F), Re: Resistant allele, East African mutation (L1014S).

Figure 6

PCR Dot blot for detection of kdr-e and kdr-w alleles. The same reactions are shown on a portion of three membranes each probed with a different sequence specific oligonucleotide probe, 104S specific for the kdr-e allele, 104L specific for the wild type allele or 104F specific for the kdr-w allele. Reaction products shown are, top row (left to right): no template control, homozygous wild type, homozygous kdr-e, homozygous kdr-e, bottom row (left to right): heterozygous kdr-w/wild type, heterozygous kdr-w/wild type, heterozygous kdr-w/wild type, heterozygous kdr-e/wild type.