A new tool for the molecular identification of Culicoides species of the Obsoletus group: the glass slide microarray approach
A new tool for the molecular identification of Culicoides species of the Obsoletus group: the glass slide microarray approach
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DOI：
10.1111/j.1365-2915.2011.00979.x
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发表时间：
2012-03-01
影响因子：
1.9
通讯作者：
Geysen, D.
中科院分区：
文献类型：
--
作者：
DeBlauwe, I.;De Witte, J. C.;Geysen, D.
隐翅虫属（双翅目：蠓科）的库蠓属物种是蓝舌病毒血清型8（BTV 8）的潜在载体，该病毒于2006年引入西欧中部。正确的形态学物种鉴定的Obsoletus组女性是特别困难的，分子鉴定是首选的方法。在这项研究中，我们提出了一个新的分子工具的基础上探针杂交使用的DNA微阵列格式，以确定库蠓物种的Obsoletus组。本研究测定了13个库蠓属55个库蠓种的ITS1基因序列，并与GenBank中的19个库蠓ITS1基因序列一起设计了库蠓种特异性探针。使用属于11个物种的另外85份库蠓标本的扩增ITS 1序列对该测试进行了评估。微阵列测试成功地确定了所有样本（100%）的Obsoletus组，确定每个标本的物种水平组内。与现有的基于聚合酶链反应（PCR）的分子工具相比，该测试具有几个优点，包括可能对许多物种进行平行分析的能力，高灵敏度和特异性，以及低背景信号噪声。手工点样微阵列载玻片和使用检测化学使得这种替代技术对于任何具有PCR设施的诊断实验室来说都是负担得起的和可行的。
Culicoides species of the Obsoletus group (Diptera: Ceratopogonidae) are potential vectors of bluetongue virus serotype 8 (BTV 8), which was introduced into central Western Europe in 2006. Correct morphological species identification of Obsoletus group females is especially difficult and molecular identification is the method of choice. In this study we present a new molecular tool based on probe hybridization using a DNA microarray format to identify Culicoides species of the Obsoletus group. The internal transcribed spacer 1 (ITS1) gene sequences of 55 Culicoides belonging to 13 different species were determined and used, together with 19 Culicoides ITS1 sequences sourced from GenBank, to design species-specific probes for the microarray test. This test was evaluated using the amplified ITS1 sequences of another 85 Culicoides specimens, belonging to 11 species. The microarray test successfully identified all samples (100%) of the Obsoletus group, identifying each specimen to species level within the group. This test has several advantages over existing polymerase chain reaction (PCR)-based molecular tools, including possible capability for parallel analysis of many species, high sensitivity and specificity, and low background signal noise. Hand-spotting of the microarray slide and the use of detection chemistry make this alternative technique affordable and feasible for any diagnostic laboratory with PCR facilities.