科研之友
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摘要
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.
- 出版日期2015-7
- 单位哈尔滨工业大学; 中国科学院海洋研究所
