Verhaeghen Katrijn, Van Bortel Wim, Roelants Patricia, Backeljau Thierry, Coosemans Marc
Departement of Parasitology, Prins Leopold Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerp, Belgium.
Malar J. 2006 Feb 22;5:16. doi: 10.1186/1475-2875-5-16.
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.
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.
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.
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.
对病媒抗杀虫剂情况进行适当监测是疟疾控制项目中杀虫剂使用规划和评估的一个重要组成部分。冈比亚按蚊和阿拉伯按蚊这两种疟疾媒介由于一种导致神经系统敏感性降低的机制（即击倒抗性，简称kdr），已对拟除虫菊酯类杀虫剂产生了抗性。在冈比亚按蚊和阿拉伯按蚊中，副型钠通道中的两种不同替代与kdr有关，一种是在西非常见的L1014F替代，另一种是在肯尼亚发现的L1014S替代。需要两种不同的等位基因特异性聚合酶链反应（AS-PCR）来检测这些已知的kdr突变。然而，这些AS-PCR检测依赖于单核苷酸多态性错配，这可能导致结果不可靠。
本文介绍了一种基于荧光共振能量转移/熔解曲线分析（FRET/MCA）检测冈比亚按蚊和阿拉伯按蚊击倒抗性的新检测方法，并与现有检测方法进行比较。
新的FRET/MCA方法具有在一次检测中同时检测两种kdr等位基因的重要优势。此外，结果表明FRET/MCA比现有的AS-PCR检测更可靠、更灵敏，并且能够检测新的基因型。通过使用该技术，首次在乌干达的阿拉伯按蚊标本中发现了东非kdr突变（L1014S）。此外，在乌干达的冈比亚按蚊中报告了一种新的kdr基因型，其中有四只冈比亚按蚊同时拥有西非（L1014F）和东非（L1014S）kdr等位基因。
在同一地理区域同时存在两种kdr突变表明需要一种可靠的检测方法，能够在一次检测中同时检测这两种突变。因此，这种基于FRET/MCA的新检测方法将改进对冈比亚按蚊和阿拉伯按蚊中kdr频率的筛查。
