Identification of field caught Anopheles gambiae s.s. and Anopheles arabiensis by TaqMan single nucleotide polymorphism genotyping
Identification of field caught Anopheles gambiae s.s. and Anopheles arabiensis by TaqMan single nucleotide polymorphism genotyping
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DOI：
10.1186/1475-2875-6-23
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发表时间：
2007-02-27
期刊：
影响因子：
3
通讯作者：
Vulule, John M.
中科院分区：
文献类型：
--
作者：
Walker, Edward D.;Thibault, Alisha R.;Vulule, John M.
背景：冈比亚按蚊的鉴定。和野外采集的冈比亚按蚊中的阿拉伯按蚊。在基础研究和应用研究以及业务控制方案中往往是必要的。目前公认的方法包括使用标准的聚合酶链式反应扩增基因组3‘28S到5’基因间隔区的核糖体DNA(RDNA)，并在琼脂糖凝胶上目视确认预期大小的扩增产物。本文介绍了一种基于TaqMan(TM)单核苷酸多态(SNP)基因分型的自动化定量聚合酶链式反应方法的建立和评价。方法：标准的聚合酶链式反应和TaqMan单核苷酸多态(SNP)基因分型方法，用新设计的引物和荧光标记的探针杂交到两种AN的互补rDNA序列。冈比亚亚种或者是一个。Arabiens，进行了三个实验，涉及野外采集的An.冈比亚亚种来自肯尼亚西部，并确定了实验室菌株。结果：TaqMan SNP基因分型的反应成功率、敏感性、种属特异性与标准PCR相当。在一项广泛的实地研究中，3,041只中只有29只(0.95%)被TaqMan确定为杂交种(即具有两个物种的rDNA序列)，然而，除了一只外，所有的都是An。结论：TaqMan SNP基因分型是一种敏感、快速的检测方法。冈比亚亚种还有一个。Arabisis，成功率高，结果特异，与标准的聚合酶链式反应方法一致。
Background: Identification of Anopheles gambiae s.s. and Anopheles arabiensis from field-collected Anopheles gambiae s.l. is often necessary in basic and applied research, and in operational control programmes. The currently accepted method involves use of standard polymerase chain reaction amplification of ribosomal DNA (rDNA) from the 3' 28S to 5' intergenic spacer region of the genome, and visual confirmation of amplicons of predicted size on agarose gels, after electrophoresis. This report describes development and evaluation of an automated, quantitative PCR method based upon TaqMan(TM) single nucleotide polymorphism (SNP) genotyping.Methods: Standard PCR, and TaqMan SNP genotyping with newly designed primers and fluorophore-labeled probes hybridizing to sequences of complementary rDNA specific for either An. gambiae s.s. or An. arabiensis, were conducted in three experiments involving field-collected An. gambiae s.l. from western Kenya, and defined laboratory strains. DNA extraction was from a single leg, sonicated for five minutes in buffer in wells of 96-well PCR plates.Results: TaqMan SNP genotyping showed a reaction success rate, sensitivity, and species specificity comparable to that of standard PCR. In an extensive field study, only 29 of 3,041 (0.95%) were determined to be hybrids by TaqMan (i.e., having rDNA sequences from both species), however, all but one were An. arabiensis by standard PCR, suggesting an acceptably low (ca. 1%) error rate for TaqMan genotyping in mistakenly identifying species hybrids.Conclusion: TaqMan SNP genotyping proved to be a sensitive and rapid method for identification of An. gambiae s.l. and An. arabiensis, with a high success rate, specific results, and congruence with the standard PCR method.