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首页> 外文期刊>Harmful Algae >Simultaneous detection of harmful algae by multiple polymerase chain reaction coupled with reverse dot blot hybridization
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Simultaneous detection of harmful algae by multiple polymerase chain reaction coupled with reverse dot blot hybridization

机译：多重聚合酶链反应与反向斑点杂交杂交同时检测有害藻类

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摘要

Harmful algal blooms (HABs) caused by microscopic algae present a threat to human health, ecosystem, fishery, tourism, and aquaculture worldwide. HAB warning and monitoring projects require a simple and rapid method for accurate parallel identification of causative algae. This study presents a useful method for simultaneous detection of harmful algae by multiple PCR coupled with reverse dot blot hybridization (MPCRDBH). A variety of probes, including positive, negative, and specific, were first developed by sequencing and consequent sequence analysis of large subunit rDNA D1-D2 from target species and used for specificity test by blot hybridization. The MPCRDBH assay mainly included five steps: (1) microalgal DNA isolation; (2) amplification and labeling of target DNA by multiple PCR; (3) probe tailing and fixation onto positively charged nylon membrane; (4) reverse dot blot hybridization; and (5) hybridization signal recognition by naked eyes. The reverse dot blot hybridization conditions were optimized, and the appropriate parameters were as follows: ultraviolet cross-linking time, 0.5 min; probe density, 2μM; Dig-labeled PCR product density, 200 ng; hybridization time and temperature, 2 h and 42 ℃; and washing time and temperature, 2×5 min and 47 ℃. Sensitivity tests showed that MPCRDBH demonstrated a detection limit of 0.6 cell. MPCRDBH recovered all target species and was not affected by background DNA. MPCRDBH also demonstrated a stable detection performance for fixative (acidic Lugol's solution)-preserved samples over 30 d using simulated field samples. MPCRDBH applicability was assessed and proven effective for parallel detection of target microalgae in the field samples. The developed MPCRDBH exhibited a simple membrane-based DNA array preparation and hybridization signal recognition compared with other current DNA arrays. The assay presented in this study is specific and sensitive for parallel detection of microalgae, with stable performance. Therefore, this assay is promising for field monitoring of natural samples.
机译：微观藻类造成的有害藻华（HAB）对全球人类健康，生态系统，渔业，旅游业和水产养殖业构成威胁。 HAB警告和监视项目需要一种简单而快速的方法来准确并行地识别病原藻。这项研究提出了一种有用的方法，可通过多重PCR与反向斑点杂交（MPCRDBH）结合同时检测有害藻类。首先通过对目标物种的大亚基rDNA D1-D2进行测序和随后的序列分析，开发出包括阳性，阴性和特异性在内的多种探针，并通过印迹杂交将其用于特异性测试。 MPCRDBH分析主要包括五个步骤：（1）微藻DNA的分离； （2）通过多重PCR扩增和标记靶DNA； （3）将探针拖尾并固定在带正电的尼龙膜上； （4）反向斑点杂交； （5）肉眼识别杂交信号。优化了反向斑点杂交的条件，合适的参数如下：紫外交联时间0.5min；紫外交联时间0.5min。探针密度2μM; Dig标记的PCR产物密度为200 ng；杂交时间和温度分别为2 h和42℃；洗涤时间和温度为2×5分钟，温度为47℃。敏感性测试表明，MPCRDBH的检出限为0.6个细胞。 MPCRDBH回收了所有目标物种，并且不受背景DNA的影响。 MPCRDBH还显示了在30天内使用模拟野外样品对固定剂（酸性Lugol溶液）保存的样品具有稳定的检测性能。评估了MPCRDBH的适用性，并证明了其对田间样品中目标微藻的平行检测有效。与其他目前的DNA阵列相比，开发的MPCRDBH展示了一种简单的基于膜的DNA阵列制备方法和杂交信号识别功能。在这项研究中提出的测定法对微藻类的平行检测具有特异性和敏感性，且性能稳定。因此，该测定法有望用于天然样品的现场监测。

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  • 来源
    《Harmful Algae》 |2014年第5期|9-19|共11页
  • 作者

    Chun Yun Zhang; Guo Fu Chen; Yang Liu; Yuan Yuan Wang; Zhong Xu; Bao Yu Zhang; Guang Ce Wang;

  • 作者单位

    College of Oceanology, Harbin Institute of Technology (Weihai), Weihai 264209, PR China;

    School of Marine Science and Technology, Harbin Institute of Technology (Weihai), Wenhua West Road, 2, Weihai, Shandong Province, PR China;

    College of Oceanology, Harbin Institute of Technology (Weihai), Weihai 264209, PR China;

    College of Oceanology, Harbin Institute of Technology (Weihai), Weihai 264209, PR China;

    College of Oceanology, Harbin Institute of Technology (Weihai), Weihai 264209, PR China;

    Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, PR China;

    Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, PR China;

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  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

    Harmful algal blooms; LSU rDNA; Multiple PCR; Reverse dot blot hybridization;

    机译：有害的藻华;LSU rDNA;多重PCR;反向斑点杂交;

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