Detection of knockdown resistance (kdr) mutations in Anopheles gambiae: a comparison of two new high-throughput assays with existing methods.
Detection of knockdown resistance (kdr) mutations in Anopheles gambiae: a comparison of two new high-throughput assays with existing methods.
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DOI：
10.1186/1475-2875-6-111
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发表时间：
2007-08-13
期刊：
影响因子：
3
通讯作者：
Field, Linda M.
中科院分区：
文献类型：
--
作者：
Bass, Chris;Nikou, Dimitra;Donnelly, Martin J.;Williamson, Martin S.;Ranson, Hilary;Ball, Amanda;Vontas, John;Field, Linda M.
击倒抗性（kdr）是许多昆虫物种对拟除虫菊酯杀虫剂产生抗性的一种明确机制，是由拟除虫菊酯靶位点对位型钠通道的点突变引起的。冈比亚按蚊（非洲最重要的疟疾病媒）中 kdr 突变的存在已通过多种分子技术进行监测。然而，很少有报告比较这些不同测定的性能。在这项研究中，开发了两种新的高通量检测方法，并与四种已建立的技术进行了比较。开发了基于 1) TaqMan 探针和 2) 高分辨率熔解 (HRM) 分析的荧光测定法，用于检测 An 中的 kdr 等位基因。冈比亚。之前报道的四种 kdr 检测技术、等位基因特异性聚合酶链反应 (AS-PCR)、加热寡核苷酸连接测定 (HOLA)、序列特异性寡核苷酸探针 - 酶联免疫吸附测定 (SSOP-ELISA) 和 PCR 点印迹也进行了优化。然后，在 96 个单一昆虫样本（包括各种 kdr 基因型和非洲按蚊物种）的盲法基因分型试验中，比较了所有六种检测的灵敏度和特异性。每个测定的相对优点是根据基因分型试验的表现、每个方案的长度/难度、成本（资本支出和消耗品成本）和安全性（危险化学品的要求）来评估的。实时 TaqMan 检测既是最灵敏的（失败反应的数量最少）也是最特异的（错误评分的数量最少）。将 TaqMan 测定调整为使用 PCR 仪并使用荧光计进行终点测量显示灵敏度和特异性略有下降。 HRM 最初给出了有希望的结果，但对 DNA 质量和数量更加敏感，因此显示出更高的失败率和错误分数。 AS-PCR、SSOP-ELISA、PCR Dot Blot 和 HOLA 的敏感性和特异性相当相似，有少量失败和错误评分。每种检测的盲法基因分型试验结果表明，在需要最大灵敏度和特异性的情况下，TaqMan 实时检测是首选方法。然而，这种测定的成本，特别是在初始资本支出方面，高于其他一些方法。使用 PCR 仪和荧光计进行的 TaqMan 检测几乎与实时检测一样灵敏，并且可以节省资本支出。如果价格是分析选择的主要因素，那么 AS-PCR、SSOP-ELISA 和 HOLA 都是合理的替代方案，其中 SSOP-ELISA 方法具有最高的通量。
Knockdown resistance (kdr) is a well-characterized mechanism of resistance to pyrethroid insecticides in many insect species and is caused by point mutations of the pyrethroid target site the para-type sodium channel. The presence of kdr mutations in Anopheles gambiae, the most important malaria vector in Africa, has been monitored using a variety of molecular techniques. However, there are few reports comparing the performance of these different assays. In this study, two new high-throughput assays were developed and compared with four established techniques. Fluorescence-based assays based on 1) TaqMan probes and 2) high resolution melt (HRM) analysis were developed to detect kdr alleles in An. gambiae. Four previously reported techniques for kdr detection, Allele Specific Polymerase Chain Reaction (AS-PCR), Heated Oligonucleotide Ligation Assay (HOLA), Sequence Specific Oligonucleotide Probe – Enzyme-Linked ImmunoSorbent Assay (SSOP-ELISA) and PCR-Dot Blot were also optimized. The sensitivity and specificity of all six assays was then compared in a blind genotyping trial of 96 single insect samples that included a variety of kdr genotypes and African Anopheline species. The relative merits of each assay was assessed based on the performance in the genotyping trial, the length/difficulty of each protocol, cost (both capital outlay and consumable cost), and safety (requirement for hazardous chemicals). The real-time TaqMan assay was both the most sensitive (with the lowest number of failed reactions) and the most specific (with the lowest number of incorrect scores). Adapting the TaqMan assay to use a PCR machine and endpoint measurement with a fluorimeter showed a slight reduction in sensitivity and specificity. HRM initially gave promising results but was more sensitive to both DNA quality and quantity and consequently showed a higher rate of failure and incorrect scores. The sensitivity and specificity of AS-PCR, SSOP-ELISA, PCR Dot Blot and HOLA was fairly similar with a small number of failures and incorrect scores. The results of blind genotyping trials of each assay indicate that where maximum sensitivity and specificity are required the TaqMan real-time assay is the preferred method. However, the cost of this assay, particularly in terms of initial capital outlay, is higher than that of some of the other methods. TaqMan assays using a PCR machine and fluorimeter are nearly as sensitive as real-time assays and provide a cost saving in capital expenditure. If price is a primary factor in assay choice then the AS-PCR, SSOP-ELISA, and HOLA are all reasonable alternatives with the SSOP-ELISA approach having the highest throughput.
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影响因子：
2.6
作者：
Fanello, C;Petrarca, V;Coluzzi, M
通讯作者：
Coluzzi, M
DOI：
10.4269/ajtmh.2005.72.155
发表时间：
2005-02-01
影响因子：
3.3
作者：
Alifrangis, M;Enosse, S;Bygjerg, IBC
通讯作者：
Bygjerg, IBC
DOI：
10.4269/ajtmh.1995.52.377
发表时间：
1995-05-01
影响因子：
3.3
作者：
CHOI, HW;BREMAN, JG;SEXTON, JD
通讯作者：
SEXTON, JD
影响因子：
3.8
作者：
Krypuy M;Newnham GM;Thomas DM;Conron M;Dobrovic A
通讯作者：
Dobrovic A
DOI：
10.1016/s1050-3862(98)00019-9
发表时间：
1999-02-01
期刊：
GENETIC ANALYSIS-BIOMOLECULAR ENGINEERING
影响因子：
--
作者：
Livak, KJ
通讯作者：
Livak, KJ