一种用于检测吡虫啉抗性埃及伊蚊击倒抗性突变 V1016G 和 F1534C 的多重 PCR 方法。
A multiplex PCR for detection of knockdown resistance mutations, V1016G and F1534C, in pyrethroid-resistant Aedes aegypti.
机构信息
Graduate School, Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand.
Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand.
出版信息
Parasit Vectors. 2017 Oct 10;10(1):465. doi: 10.1186/s13071-017-2416-x.
BACKGROUND
Mutation of the voltage-gated sodium channel (VGSC) gene, or knockdown resistance (kdr) gene, is an important resistance mechanism of the dengue vector Aedes aegypti mosquitoes against pyrethroids. In many countries in Asia, a valine to glycine substitution (V1016G) and a phenylalanine to cysteine substitution (F1534C) are common in Ae. aegypti populations. The G1016 and C1534 allele frequencies have been increasing in recent years, and hence there is a need to have a simple and inexpensive tool to monitor the alleles in large scale.
METHODS
A multiplex PCR to detect V1016G and F1534C mutations has been developed in the current study. This study utilized primers from previous studies for detecting the mutation at position 1016 and newly designed primers to detect variants at position 1534. The PCR conditions were validated and compared with DNA sequencing using known kdr mutant laboratory strains and field collected mosquitoes. The efficacy of this method was also compared with allele-specific PCR (AS-PCR).
RESULTS
The results of our multiplex PCR were in complete agreement with sequencing data and better than the AS-PCR. In addition, the efficiency of two non-toxic DNA staining dyes, Ultrapower™ and RedSafe™, were evaluated by comparing with ethidium bromide (EtBr) and the results were satisfactory.
CONCLUSIONS
Our multiplex PCR method is highly reliable and useful for implementing vector surveillance in locations where the two alleles co-occur.
背景
电压门控钠离子通道（VGSC）基因突变或击倒抗性（kdr）基因突变是登革热传播媒介埃及伊蚊对拟除虫菊酯产生抗药性的重要机制。在亚洲许多国家，埃及伊蚊种群中常见缬氨酸到甘氨酸取代（V1016G）和苯丙氨酸到半胱氨酸取代（F1534C）。近年来，G1016 和 C1534 等位基因频率一直在增加，因此需要有一种简单且廉价的工具来大规模监测这些等位基因。
方法
本研究开发了一种用于检测 V1016G 和 F1534C 突变的多重 PCR。本研究利用先前研究中用于检测 1016 位突变的引物和新设计的引物来检测 1534 位的变体。验证了 PCR 条件，并与使用已知 kdr 突变实验室品系和现场采集的蚊子进行 DNA 测序进行了比较。还比较了该方法与等位基因特异性 PCR（AS-PCR）的效果。
结果
我们的多重 PCR 结果与测序数据完全一致，且优于 AS-PCR。此外，通过与溴化乙锭（EtBr）比较，评估了两种无毒 DNA 染色染料 Ultrapower™和 RedSafe™的效率，结果令人满意。
结论
我们的多重 PCR 方法非常可靠且可用于在两个等位基因同时存在的地方实施媒介监测。
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