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首页> 外文期刊>International Journal of Environmental Research and Public Health >Molecular Tools for the Selective Detection of Nine Diatom Species Biomarkers of Various Water Quality Levels
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Molecular Tools for the Selective Detection of Nine Diatom Species Biomarkers of Various Water Quality Levels

机译：选择性检测不同水质水平的9种硅藻物种生物标志物的分子工具

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Our understanding of the composition of diatom communities and their response to environmental changes is currently limited by laborious taxonomic identification procedures. Advances in molecular technologies are expected to contribute more efficient, robust and sensitive tools for the detection of these ecologically relevant microorganisms. There is a need to explore and test phylogenetic markers as an alternative to the use of rRNA genes, whose limited sequence divergence does not allow the accurate discrimination of diatoms at the species level. In this work, nine diatom species belonging to eight genera, isolated from epylithic environmental samples collected in central Italy, were chosen to implement a panel of diatoms covering the full range of ecological status of freshwaters. The procedure described in this work relies on the PCR amplification of specific regions in two conserved diatom genes, elongation factor 1-a (eEF1-a) and silicic acid transporter (SIT), as a first step to narrow down the complexity of the targets, followed by microarray hybridization experiments. Oligonucleotide probes with the potential to discriminate closely related species were designed taking into account the genetic polymorphisms found in target genes. These probes were tested, refined and validated on a small-scale prototype DNA chip. Overall, we obtained 17 highly specific probes targeting eEF1-a and SIT, along with 19 probes having lower discriminatory power recognizing at the same time two or three species. This basic array was validated in a laboratory setting and is ready for tests with crude environmental samples eventually to be scaled-up to include a larger panel of diatoms. Its possible use for the simultaneous detection of diatoms selected from the classes of water quality identified by the European Water Framework Directive is discussed.
机译：目前我们对硅藻群落的组成及其对环境变化的反应的了解目前受到繁琐的生物分类鉴定程序的限制。分子技术的进步有望为检测这些与生态相关的微生物提供更有效，更强大和更灵敏的工具。需要探索和测试系统发育标记，以替代rRNA基因的使用，rRNA基因的有限序列差异使得无法在物种水平上准确区分硅藻。在这项工作中，从意大利中部采集的火山灰环境样品中分离出属于8个属的9个硅藻物种，以实施一组涵盖整个淡水生态状况的硅藻。这项工作中描述的程序依赖于PCR扩增两个保守的硅藻基因中的特定区域，即延伸因子1-a（eEF1-a）和硅酸转运蛋白（SIT），这是缩小靶标复杂性的第一步，然后进行微阵列杂交实验。考虑到在靶基因中发现的遗传多态性，设计出可能区分密切相关物种的寡核苷酸探针。这些探针在小规模的原型DNA芯片上进行了测试，改进和验证。总体而言，我们获得了针对eEF1-a和SIT的17种高特异性探针，以及同时识别两种或三种物种的19种具有较低鉴别力的探针。该基本阵列已在实验室环境中得到验证，并准备用于粗环境样品的测试，最终将其扩大规模以包括更大的硅藻板。讨论了其在同时检测选自欧洲水框架指令确定的水质类别中的硅藻的可能用途。

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