Djogbénou Luc S, Assogba Benoît, Essandoh John, Constant Edi A V, Makoutodé Michel, Akogbéto Martin, Donnelly Martin J, Weetman David
Institut Regional de Santé Publique de Ouidah/Université d'Abomey-Calavi, Cotonou, Benin.
Department of Vector Biology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, UK.
Malar J. 2015 Dec 18;14:507. doi: 10.1186/s12936-015-1026-3.
Identification of variation in Ace-1 copy number and G119S mutation genotype from samples of Anopheles gambiae and Anopheles coluzzii across West Africa are important diagnostics of carbamate and organophosphate resistance at population and individual levels. The most widespread and economical method, PCR-RFLP, suffers from an inability to discriminate true heterozygotes from heterozygotes with duplication.
In addition to PCR-RFLP, in this study three different molecular techniques were applied on the same mosquito specimens: TaqMan qPCR, qRTPCR and ddPCR. To group heterozygous individuals recorded from the PCR-RFLP analysis into different assumptive genotypes K-means clustering was applied on the Z-scores of data obtained from both the TaqMan and ddPCR methods. The qRTPCR analysis was used for absolute quantification of copy number variation.
The results indicate that most heterozygotes are duplicated and that G119S mutation must now be regarded as a complex genotype ranging from primarily single-copy susceptible Glycine homozygotes to balanced and imbalanced heterozygotes, and multiply-amplified resistant Serine allele homozygotes. Whilst qRTPCR-based gene copy analysis suffers from some imprecision, it clearly illustrates differences in copy number among genotype groups identified by TaqMan or ddPCR. Based on TaqMan method properties, and by coupling TaqMan and ddPCR methods simultaneously on the same type of mosquito specimens, it demonstrated that the TaqMan genotype assays associated with the K-means clustering algorithm could provide a useful semi-quantitative estimate method to investigate the level of allele-specific duplication in mosquito populations.
Ace-1 gene duplication is evidently far more complex in An. gambiae and An. coluzzii than the better-studied mosquito Culex quinquefasciatus, which consequently can no longer be considered an appropriate model for prediction of phenotypic consequences. These require urgent further evaluation in Anopheles. To maintain the sustained effectiveness carbamates and organophosphates as alternative products to pyrethroids for malaria vector control, monitoring of duplicated resistant alleles in natural populations is essential to guide the rational use of these insecticides.
在西非各地，从冈比亚按蚊和科氏按蚊样本中鉴定Ace-1拷贝数变异和G119S突变基因型，是在种群和个体水平上诊断对氨基甲酸酯类和有机磷酸酯类抗性的重要方法。最广泛且经济的方法——聚合酶链反应-限制性片段长度多态性（PCR-RFLP），存在无法区分真正的杂合子与具有重复的杂合子的问题。
在本研究中，除了PCR-RFLP外，还对相同的蚊子标本应用了三种不同的分子技术：TaqMan定量聚合酶链反应（qPCR）、定量逆转录聚合酶链反应（qRTPCR）和数字滴液聚合酶链反应（ddPCR）。为了将从PCR-RFLP分析中记录的杂合个体分组为不同的假定基因型，对从TaqMan和ddPCR方法获得的数据的Z分数应用K均值聚类。qRTPCR分析用于拷贝数变异的绝对定量。
结果表明，大多数杂合子是重复的，并且G119S突变现在必须被视为一种复杂的基因型，范围从主要是单拷贝易感甘氨酸纯合子到平衡和不平衡的杂合子，以及多重扩增的抗性丝氨酸等位基因纯合子。虽然基于qRTPCR的基因拷贝分析存在一些不精确性，但它清楚地说明了通过TaqMan或ddPCR鉴定的基因型组之间拷贝数的差异。基于TaqMan方法的特性，并通过在同一类型的蚊子标本上同时结合TaqMan和ddPCR方法，证明与K均值聚类算法相关的TaqMan基因型测定可以提供一种有用的半定量估计方法，以研究蚊子种群中等位基因特异性重复的水平。
在冈比亚按蚊和科氏按蚊中，Ace-1基因重复显然比研究得更好的致倦库蚊复杂得多，因此致倦库蚊不能再被视为预测表型后果的合适模型。这些需要在按蚊中进行紧急进一步评估。为了维持氨基甲酸酯类和有机磷酸酯类作为拟除虫菊酯类替代产品用于疟疾媒介控制的持续有效性，监测自然种群中重复的抗性等位基因对于指导这些杀虫剂的合理使用至关重要。
