基于RPA-LFD技术的塔玛亚历山大藻快速检测方法的建立
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Q949.2

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国家重点研发计划(2022YFC2601301)


Establishment of detection method for Alexandrium tamarense by recombinase polymerase amplification coupled with lateral flow dipstick
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    摘要:

    为了满足塔玛亚历山大藻(Alexandrium tamarense)在野外环境中快速检测的需求,以塔玛亚历山大藻内转录间隔区(Internal transcribed spacer, ITS)序列为靶标,设计了特异性引物和探针,将重组酶聚合酶扩增技术(Recombinase polymerase amplification, RPA)与横向流动试纸条(Lateral flow dipstick, LFD)相结合(RPA-LFD),实现了扩增结果的可视化。结果显示,RPA最佳反应条件为42 ℃、15 min,LFD检测时间为3 min。研究结果表明,基于RPA-LFD技术开发的塔玛亚历山大藻检测方法具有高度特异性,与常见藻类无交叉反应,能够特异性检出塔玛亚历山大藻。该方法对塔玛亚历山大藻质粒和细胞的检出限分别达到1 copies/μL和1 cells/L,较常规PCR提高了2个数量级。从特异性、灵敏度、便携性及时效性等方面综合评估,该方法可为实现塔玛亚历山大藻藻华的早期预警以及提升口岸对物种入侵的检测效率提供支持。

    Abstract:

    To meet the need for rapid detection of Alexandrium tamarense in field environments, it is imperative to develop a real-time method with high sensitivity and specificity. In this study, a visual method referred to as recombinase polymerase amplification coupled with lateral flow dipstick (RPA-LFD) was developed for rapid detection. The primers for RPA and the detection probe for LFD were designed to specifically target the internal transcribed spacer of A. tamarense by molecular cloning and multiple alignments of the related sequences. The results showed that the successful amplification for RPA could be achieved at optimal conditions of 42 °C and 15 min, and enough products were visually detected by the naked eye through an LFD after an automatic chromatography for 3 min at room temperature. The research indicated that the RPA-LFD exhibited exceptional specificity for A. tamarense and displayed no cross-reactivity with other common algae. The detection limits were as low as 1 copies/μL for plasmid DNA and 1 cells/L for intact cells, representing a 100-fold increase in sensitivity compared to conventional PCR methods. In conclusion, the developed RPA-LFD assay enables rapid detection of A. tamarense-induced HABs while enhancing invasive species screening efficiency at ports.

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吴文静,孙鹤宸,康伟,胡健伟,高倩,王翠华.基于RPA-LFD技术的塔玛亚历山大藻快速检测方法的建立[J].上海海洋大学学报,2025,34(5):956-967.
WU Wenjing, SUN Hechen, KANG Wei, HU Jianwei, GAO Qian, WANG Cuihua. Establishment of detection method for Alexandrium tamarense by recombinase polymerase amplification coupled with lateral flow dipstick[J]. Journal of Shanghai Ocean University,2025,34(5):956-967.

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  • 收稿日期:2025-04-29
  • 最后修改日期:2025-07-19
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  • 在线发布日期: 2025-09-25
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