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首页> 外文期刊>Harmful Algae >Establishment and application of hyperbranched rolling circle amplification coupled with lateral flow dipstick for the sensitive detection of Karenia mikimotoi
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Establishment and application of hyperbranched rolling circle amplification coupled with lateral flow dipstick for the sensitive detection of Karenia mikimotoi

机译：超支化滚环扩增法与侧向量油尺的建立及其对米克雷克氏菌的灵敏检测

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摘要

The dinoflagellate Karenia mikimotoi is a noxious and harmful algal bloom (HAB)-forming microalga. Establishing a rapid, accurate, and sensitive method of detecting this harmful alga is necessary to provide warnings of imminent HABs through field monitoring. Here, an isothermal amplification technique combined with a rapid analytical method for nucleic acid-based amplified products, i.e., hyperbranched rolling circle amplification (HRCA) coupled with lateral flow dipstick (LFD), hereafter denoted as HRCA-LFD, was established to detect K. mikimotoi. The HRCA-LFD assay relied on a padlock probe (PLP) targeting DNA template and an LFD probe targeting PLP. The sequenced internal transcribed spacer of K. mikimotoi through molecular cloning was used as the target of PLP. The optimized HRCA conditions was determined to be as follows: PLP concentration, 20 pM; ligation temperature, 65 degrees C; ligation time, 10 min; amplification temperature, 61 degrees C; and amplification time, 30 min. The developed HRCA-LFD assay was specific for K. mikimotoi, displaying no cross-reactivity with other common microalgae. Sensitivity-comparison tests indicated that HRCA-LFD assay was 100-fold more sensitive than PCR, with a detection limit of 0.1 cell mL(-1) when used to analyze spiked field samples. The analysis with field samples also indicated that HRCA-LFD assay was suitable for samples with a target cell density range of 1-1000 cells mL(-1). All of these results suggested that HRCA-LFD assay is an alternative method for the sensitive and reliable detection of K. mikimotoi from marine water samples.
机译：鞭毛藻（Kenonia mikimotoi）是形成有害有害藻华（HAB）的微藻。建立一种快速，准确和灵敏的检测有害藻类的方法对于通过现场监视提供即将发生的HAB的警告是必要的。在这里，建立了一种等温扩增技术和一种快速分析方法相结合的基于核酸的扩增产物，即超支化滚环扩增（HRCA）和侧向量油尺（LFD），以下称为HRCA-LFD，用于检测K mikimotoi。 HRCA-LFD分析依赖于靶向DNA模板的挂锁探针（PLP）和靶向PLP的LFD探针。通过分子克隆测序的内部转录的K. mikimotoi间隔子用作PLP的靶标。确定最佳的HRCA条件如下：PLP浓度为20 pM；结扎温度65℃；结扎时间，10分钟；放大温度为61摄氏度；扩增时间为30分钟。研发的HRCA-LFD分析法对mikimotoi菌具有特异性，与其他常见微藻没有交叉反应。灵敏度比较测试表明，HRCA-LFD测定的灵敏度比PCR高100倍，当用于分析加标的野外样品时，检出限为0.1细胞mL（-1）。对现场样品的分析还表明，HRCA-LFD分析适用于目标细胞密度范围为1-1000个细胞mL（-1）的样品。所有这些结果表明，HRCA-LFD测定法是从海水样品中灵敏可靠地检测K. mikimotoi的另一种方法。

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  • 来源
    《Harmful Algae》 |2019年第4期|151-160|共10页
  • 作者

    Zhang Chunyun; Chen Guofu; Wang Yuanyuan; Zhou Jin; Li Chenghua;

  • 作者单位

    Ningbo Univ, Sch Marine Sci, Ningbo 315211, Zhejiang, Peoples R China|Harbin Inst Technol Weihai, Sch Marine Sci & Technol, Wenhua West Rd 2, Weihai 264209, Shandong, Peoples R China;

    Harbin Inst Technol Weihai, Sch Marine Sci & Technol, Wenhua West Rd 2, Weihai 264209, Shandong, Peoples R China;

    Harbin Inst Technol Weihai, Sch Marine Sci & Technol, Wenhua West Rd 2, Weihai 264209, Shandong, Peoples R China;

    Tsinghua Univ, Grad Sch Shenzhen, Div Ocean Sci & Technol, Shenzhen 518055, Peoples R China;

    Ningbo Univ, Sch Marine Sci, Ningbo 315211, Zhejiang, Peoples R China;

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  • 正文语种 eng
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  • 关键词

    Karenia mikimotoi; LSU rDNA; Hyperbranched rolling circle amplification; Lateral flow dipstick; Detection;

    机译：三叶Karenia;LSU rDNA;超支化滚环扩增;侧向量油尺;检测;

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