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首页> 外文期刊>Environmental Science and Pollution Research >Application of reverse dot blot hybridization to simultaneous detection and identification of harmful algae
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Application of reverse dot blot hybridization to simultaneous detection and identification of harmful algae

机译：反向斑点杂交技术在有害藻类同时检测和鉴定中的应用

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Warning and monitoring projects of harmful algal blooms require simple and rapid methods for simultaneous and accurate detection and identification of causative algae present in the environmental samples. Here, reverse dot blot hybridization (RDBH) was employed to simultaneously detect several harmful algae by using five representative bloom-forming microalgae along the Chinese coast. A set of specific probes for RDBH were developed by PCR, cloning, and sequencing of the internal transcribed spacer (ITS), alignment analysis, and probe design. Each probe was oligo (dT)-tailed and spotted onto positively charged nylon membrane to make up a low-density oligonucleotide array. Universal primers designed within the conserved regions were used to amplify the ITS sequences by using genomic DNA of target as templates. The digoxigenin (Dig)-labeled PCR products were denatured and then hybridized to the oligonucleotide array. The array produced a unique hybridization pattern for each target species differentiating them from each other. The preparations of oligonucleotide array and hybridization conditions were optimized. The developed RDBH demonstrated a detection limit up to 10 cells. The detection performance of RDBH was relatively stable and not affected by non-target species and the fixation time of target species over at least 30 days. The RDBH could recover all the target species from the simulated field samples and target species confirmed by the subsequent microscopy examination in the environmental samples. These results indicate that RDBH can be a new technical platform for parallel discrimination of harmful algae and is promising for environmental monitoring of these microorganisms.
机译：有害藻华的预警和监测项目需要简单，快速的方法，以便同时准确地检测和鉴定环境样品中存在的致病藻类。在这里，反向斑点杂交（RDBH）被用来通过使用中国沿海沿岸的五个代表性的形成花粉的微藻同时检测几种有害藻。通过PCR，内部转录间隔子（ITS）的克隆和测序，比对分析和探针设计，开发了一套针对RDBH的特异性探针。每个探针都经过寡聚（dT）尾处理，并点到带正电的尼龙膜上，以构成低密度寡核苷酸阵列。在保守区域内设计的通用引物用于通过以靶标的基因组DNA为模板来扩增ITS序列。使洋地黄毒苷（Dig）标记的PCR产物变性，然后与寡核苷酸阵列杂交。该阵列为每个靶标物种产生了独特的杂交模式，从而使它们彼此区分。优化了寡核苷酸阵列的制备和杂交条件。研发的RDBH展示了最多10个细胞的检测极限。 RDBH的检测性能相对稳定，并且不受非目标物种和目标物种固定时间至少30天的影响。 RDBH可以从模拟的野外样品中回收所有目标物种，并通过随后的显微镜检查在环境样品中确认目标物种。这些结果表明，RDBH可以作为平行鉴别有害藻类的新技术平台，并有望用于这些微生物的环境监测。

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