Detection of the East and West African kdr mutation in Anopheles gambiae and Anopheles arabiensis from Uganda using a new assay based on FRET/Melt Curve analysis

Abstract

Background: Appropriate monitoring of vector resistance to insecticides is an integral component of planning and evaluation of insecticide use in malaria control programmes. The malaria vectors Anopheles gambiae s.s. and Anopheles arabiensis have developed resistance to pyrethroid insecticides as a result of a mechanism conferring reduced nervous system sensitivity, better known as knockdown resistance (kdr). In An. gambiae s.s. and An. arabiensis, two different substitutions in the para-type sodium channel, a L1014F substitution common in West Africa and a L1014S replacement found in Kenya, are linked with kdr. Two different allele-specific polymerase chain reactions (AS-PCR) are needed to detect these known kdr mutations. However, these AS-PCR assays rely on a single nucleotide polymorphism mismatch, which can result in unreliable results.

Methods: Here, a new assay for the detection of knockdown resistance in An. gambiae s.s. and An. arabiensis based on Fluorescence Resonance Energy Transfer/Melt Curve analysis (FRET/MCA) is presented and compared with the existing assays.

Results: The new FRET/MCA method has the important advantage of detecting both kdr alleles in one assay. Moreover, results show that the FRET/MCA is more reliable and more sensitive than the existing AS-PCR assays and is able to detect new genotypes. By using this technique, the presence of the East African kdr mutation (L1014S) is shown for the first time in An. arabiensis specimens from Uganda. In addition, a new kdr genotype is reported in An. gambiae s.s. from Uganda, where four An. gambiae s.s. mosquitoes possess both, the West (L1014F) and East (L1014S) African kdr allele, simultaneously.

Conclusion: The presence of both kdr mutations in the same geographical region shows the necessity of a reliable assay that enables to detect both mutations in one single assay. Hence, this new assay based on FRET/MCA will improve the screening of the kdr frequencies in An. gambiae s.s. and An. arabiensis.

Figure 1

Schematic representation of the AS-PCR Agd5 and AS-PCR Agd3. Schematic representation of the AS-PCR Agd5, which detects the L1014S kdr allele (A) and the AS-PCR Agd3, which detects the L1014F kdr allele (B) [3-4].

Figure 2

Schematic representation of the FRET/MCA, which allows the detection of both (L1014S and L1014F) kdr alleles in one assay. A. The primary PCR (primers Agd1-Agd2) results in amplification of a 293 bp fragment of the para-type sodium channel gene. B. During the secondary PCR, a 121 bp fragment is amplified and is labelled with ROX as the forward primer is extended. C. After amplification, the FAM-labelled probe hybridizes, and FRET starts to occur. The donor FAM-fluorophore is excited by incident light and because the ROX-acceptor is in close proximity, the excited state energy from FAM can be transferred. D. Melt curve analysis on the probe-amplicon hybrid. Progressive increase of temperature during melt curve analysis leads, at a specific temperature, to the dissociation of the probe from the amplicon. At this point, no FRET occurs and the ROX-fluorescence will decrease. During this MCA, the change in amount of fluorescence for each probe-template hybrid was plotted against the temperature and its negative derivative appeared as a positive peak.

Figure 3

FRET/MCA of the DIIS6 region of the para-type sodium channel gene of An. gambiae s.s. and An. arabiensis plotted as the first negative derivative of the relative fluorescence unit (-d(RFU)/dT) versus temperature function. In all 3 panels, the thicker blue horizontal line denotes the threshold for background fluorescence, and the curve entirely below denotes the results for the non-template control (gray). A. Cloned sequence variants, from low to high T_m: The L1014S allele (red), the L1014F allele (blue) and the wild type L1014L allele (green). Melting peaks were identified at 49.5°C, 52°C and 57.5°C, respectively. B. An. gambiae s.s. specimens, from low to high T_m: homozygous for the L1014S allele, T_m of 49.5°C (red) ; specimens heterozygous for both kdr alleles (L1014S/L1014F allele), T_m of 51°C (dark red) ; specimens homozygous for L1014F allele, T_m of 52°C (blue) and specimens homozygous wild type, T_m of 57.5°C (green). C. An. gambiae s.s. specimens heterozygous for the L1014S/wild type (dark red) were characterised by two melting peaks of 49.5°C and 57.5°C. Heterozygous (L1014S/wild type) An. arabiensis specimens showed the same pattern.

Figure 4

PCR products obtained using the AS-PCR Agd5 adapted from Ranson et al. [4] on An. gambiae s.s. after separation on a 2% agarose gel. Lane 1, Lane 3, Lane 9: homozygous wild type mosquitoes (L1014L/L1014L); Lane 2 and Lane 8: heterozygous specimens (L1014L/L1014S); Lane 4: homozygous resistant specimen (L1014S/L1014S); Lane 5 and Lane 7: 100 bp ladder; Lane 6: specimen scored as heterozygous (L1014L/L1014S) by AS-PCR, but scored as homozygous resistant (L1014S/L1014S) by FRET/MCA.