BACKGROUND Mutation of the voltage-gated sodium channel (VGSC) gene, or knockdown resistance (kdr) gene, is an important resistance mechanism against DDT and pyrethroids for dengue vector Aedes albopictus. A phenylalanine to serine (F1534S), leucine (F1534L) and cysteine (F1534C) substitution were detected in many Ae. albopictus populations around the world, and the mutant allele frequencies have been increasing in recent years. Therefore, it is essential to establish a simple, time-saving and cost-effective procedure to monitor the alleles in large-scale studies. METHODS Based on the mutation genotypes of the 1534 locus in the kdr gene, F/F, F/S, F/C, F/L, S/S, C/C, L/L and S/C, we designed specific forward and reverse primers and optimized the reaction conditions for establishing of the allele-specific PCR(AS-PCR) detection technique. DNA sequencing in this study was taken as the gold standard, and used to determine the accuracy of AS-PCR. RESULTS The designed AS-PCR technique showed high specificity for distinguishing the mutations at the 1534 locus, as the accuracy for F/F, F/S, F/C, F/L, S/S, C/C and S/C were 100%, 95.35%, 100%, 100%, 100%, 100% and 100%, respectively. CONCLUSIONS The designed AS-PCR technique effectively distinguished individual genotypes for the mutations at the 1534 locus in the kdr gene, which could facilitate the knockdown resistance surveillance in Ae. albopictus in large-scale studies.

中文翻译：

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建立创新和可持续的PCR技术，以对登革热媒介白纹伊蚊的敲除抗性（kdr）基因的1534个基因座进行突变。

背景技术电压门控钠通道（VGSC）基因或敲低抗性（kdr）基因的突变是对登革热媒介伊蚊（Aedes albopictus）的DDT和拟除虫菊酯的重要抗性机制。在许多Ae中检测到苯丙氨酸被丝氨酸（F1534S），亮氨酸（F1534L）和半胱氨酸（F1534C）取代。近年来，世界各地的白带虫种群以及突变等位基因的频率一直在增加。因此，建立一个简单，省时且具有成本效益的程序来监测大规模研究中的等位基因至关重要。方法根据kdr基因中1534个基因座的突变基因型，分别设计F / F，F / S，F / C，F / L，S / S，C / C，L / L和S / C正向和反向引物并优化了反应条件，以建立等位基因特异性PCR（AS-PCR）检测技术。本研究中的DNA测序被作为金标准，并用于确定AS-PCR的准确性。结果设计的AS-PCR技术具有很高的特异性，可区分1534个基因座处的突变，因为F / F，F / S，F / C，F / L，S / S，C / C和S / C的准确性分别为100％，95.35％，100％，100％，100％，100％和100％。结论所设计的AS-PCR技术有效地区分了kdr基因中1534位点突变的个体基因型，这可以促进Ae的抗击倒性监视。白化病的大规模研究。C / C和S / C分别为100％，95.35％，100％，100％，100％，100％和100％。结论所设计的AS-PCR技术有效地区分了kdr基因中1534位点突变的个体基因型，这可以促进Ae的抗击倒性监视。白化病的大规模研究。C / C和S / C分别为100％，95.35％，100％，100％，100％，100％和100％。结论所设计的AS-PCR技术有效地区分了kdr基因中1534位点突变的个体基因型，这可以促进Ae的抗击倒性监视。白化病的大规模研究。