背景:疟疾仍然是拉丁美洲的重要公共卫生问题,疟疾病媒杀虫剂抗药性的发展对消除疟疾的努力构成了重大威胁。需要监测杀虫剂的敏感性和确定杀虫剂抗性的机制,以有效指导部署适当的病媒控制措施。这里,已经开发了分子测定法来筛选与来自拉丁美洲的四个疟疾载体中的电压门控钠通道(VGSC)和乙酰胆碱酯酶-1(Ace-1)基因上的杀虫剂抗性相关的突变。
方法:设计简并引物以扩增VGSC和Ace-1基因上的部分片段。野生捕获的albimanus按蚊个体(也包括历史样本和实验室菌株的个体),Darlingi按蚊,用按蚊和假性按蚊优化PCR检测方法。对所有样品进行测序以验证PCR结果,并使用观察到的独特单倍型构建每个基因的DNA比对。
结果:设计的引物成功扩增了An中的VGSC基因。albimanus,A.Darlingi,A.vestippennis和An.pseudopunctipennis,和Ace-1基因在两个An.albimanus和An.Darlingi.DNA测序显示,与冈比亚按蚊相比,在An的VGSC基因上共有29、28、21和24个单核苷酸多态性(SNPs)。albimanus(308bp),A.达林吉(311个基点),A.假端基(263bp)和安。vestipennis(254bp),分别。在Ace-1基因的459bp片段上,在An中共检测到70个SNP。在An中检测到darlingi和59个SNP。albimanus与An相比。冈比亚.在VGSC基因上检测到的SNP都是同义的。在Ace-1基因上,在三个不同的密码子上鉴定出非同义取代。所有物种均在VGSC基因上的995密码子(以前报道为1014密码子)处显示纯合野生型kdr等位基因(编码亮氨酸),但是一个样本在Ace-1基因的280密码子(以前报道为119密码子)是杂合的,编码抗性(丝氨酸)和易感(甘氨酸)氨基酸。
结论:报道了用于扩增和筛选与杀虫剂抗性相关的VGSC和Ace-1基因区域的新分子测定法。albimanus,A.Darlingi,A.vestippennis,和一个。pseudopunctipennis.这些PCR检测方法的发展在美洲疟疾病媒靶位点抗性分析方面取得了重要进展,并将进一步促进这些物种中杀虫剂抗性机制的表征。
BACKGROUND: Malaria remains an important public health problem in Latin America, and the development of insecticide resistance in malaria vectors poses a major threat to malaria elimination efforts. Monitoring of insecticide susceptibility and the determination of the mechanisms involved in insecticide resistance are needed to effectively guide the deployment of appropriate vector control measures. Here, molecular assays have been developed to screen for mutations associated with insecticide resistance on the voltage-gated sodium channel (VGSC) and acetylcholinesterase-1 (Ace-1) genes in four malaria vectors from Latin America.
METHODS: Degenerate primers were designed to amplify a partial fragment on the VGSC and Ace-1 genes. Wild-caught individuals for Anopheles albimanus (also historical samples and individuals from a laboratory strain), Anopheles darlingi, Anopheles vestitipennis and Anopheles pseudopunctipennis were used to optimize the PCR assays. All samples were sequenced to validate the PCR results and DNA alignments were constructed for each gene using the unique haplotypes observed.
RESULTS: Primers designed successfully amplified the VGSC gene in An. albimanus, An. darlingi, An. vestitipennis and An. pseudopunctipennis, and the Ace-1 gene in both An. albimanus and An. darlingi. DNA sequencing revealed that compared with Anopheles gambiae, there were a total of 29, 28, 21 and 24 single nucleotide polymorphisms (SNPs) on the VGSC gene for An. albimanus (308 bp), An. darlingi (311 bp), An. pseudopunctipennis (263 bp) and An. vestitipennis (254 bp), respectively. On the 459 bp fragment of the Ace-1 gene, a total of 70 SNPs were detected in An. darlingi and 59 SNPs were detected in An. albimanus compared with An. gambiae. The SNPs detected on the VGSC gene were all synonymous. On the Ace-1 gene, non-synonymous substitutions were identified on three different codons. All species showed the homozygous wild-type kdr allele (coding for leucine) at codon 995 (formerly reported as codon 1014) on the VGSC gene, but one sample was heterozygous at codon 280 (formerly reported as codon 119) on the Ace-1 gene, coding for both the resistant (serine) and susceptible (glycine) amino acids.
CONCLUSIONS: New molecular assays to amplify and screen the regions of the VGSC and Ace-1 genes associated with insecticide resistance are reported for An. albimanus, An. darlingi, An. vestitipennis, and An. pseudopunctipennis. The development of these PCR assays presents an important advance in the analysis of target-site resistance in malaria vectors in the Americas, and will further facilitate the characterization of insecticide resistance mechanisms in these species.