A Simple Chelex Protocol for DNA Extraction from Anopheles spp.
A Simple Chelex Protocol for DNA Extraction from Anopheles spp.
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DOI：
10.3791/3281
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发表时间：
2013-01-01
影响因子：
1.2
通讯作者：
Mharakurwa, Sungano
中科院分区：
文献类型：
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作者：
Musapa, Mulenga;Kumwenda, Taida;Mharakurwa, Sungano
疟疾流行国家越来越多地采用分子工具，对疟疾寄生虫和病媒蚊子进行有效的分型、鉴定和监测，作为其控制计划的一个组成部分（1、2、3、4、5）。为了在运筹学中可持续地建立这些精确的方法，以加强疟疾控制和消除工作，简单和负担得起的方法，以及节俭的试剂和设备要求是必不可少的（6，7，8）。本文介绍了一种从野外采集的蚊虫标本中提取疟原虫和载体DNA的简单方法，并对72只冈比亚按蚊进行了形态学鉴定。来自马查疟疾研究所附近2，000公里（2）范围内家庭卧室中用除虫菊喷雾捕获的156只蚊子。对72只冈比亚按蚊进行头、胸、腹分离。将两个切片分别置于1.5ml微量离心管中并浸没在20 μ l去离子水中。使用无菌移液管尖端，将每个蚊子切片分别均质化成在去离子水中的均匀悬浮液。从每个蚊子切片的随后匀浆中，保留10 μ l，而将另外10 μ l转移到单独的高压灭菌的1.5ml管中。通过简化的Chelex或标准盐析提取方案对单独的等分试样进行DNA提取（9，10）。所谓的盐析法之所以被广泛使用，是因为它使用高浓度的盐来代替有害的有机溶剂提取物用作PCR扩增的模板，使用靶向节肢动物线粒体烟酰胺腺嘌呤二核苷酸脱氢酶（NADH）亚基4基因（ND4）的引物来检查DNA质量（11），一种用于冈比亚按蚊同胞种鉴定的PCR（10）和一种用于恶性疟原虫感染分型的巢式PCR（12）。使用DNA质量（ND4）PCR的比较显示，93%的敏感性和82%的特异性的Chelex方法相对于已建立的盐析协议。同胞种鉴定PCR的敏感性和特异性分别为100%和78%，恶性疟原虫检测PCR的敏感性和特异性分别为92%和80%。在所有三种PCR应用中，使用Chelex或常规盐析方案给出扩增子信号的样品比例没有显著差异。Chelex方法需要三种简单的试剂和37分钟来完成，而盐析方案需要10种不同的试剂和2小时47分钟的处理时间，包括过夜步骤。我们的研究结果表明，Chelex方法与现有的盐析提取法相当，并且可以在资源有限的环境中作为一种简单且可持续的方法来替代，在这些环境中，恒定的试剂供应链通常难以维持。
Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs(1,2,3,4,5). For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential(6,7,8). Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km(2) vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 mu l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 mu l was retained while the other 10 mu l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol(9,10). The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction(9).Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality(11), a PCR for identification of Anopheles gambiae sibling species(10) and a nested PCR for typing of Plasmodium falciparum infection(12). Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.