Development of the Visual Loop-Mediated Isothermal Amplification Combined with Lateral Flow Dipstick (LAMP-LFD) Method for Rapid Detection of Vibrio harveyi in Aquatic Animals

IF 1 4区生物学 Q4 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Applied Biochemistry and Microbiology Pub Date : 2024-06-10 DOI:10.1134/S0003683824604062

S. H. Cai, Z. Chen, L. J. Sun, J. Zhong, Y. C. Huang, S. P. Yang, Y. S. Lu

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Abstract

Vibrio harveyi is a normal flora present in seawater, but in recent years, with large density aquaculture, V. harveyi has become one of the causative organisms in fish and shrimp. In this study, V. harveyi VieA gene was used as the target to design primers and establish the loop-mediated isothermal amplification (LAMP) combined with visual lateral flow test strip (LFD). The optimal reaction conditions were optimized as 60°C, 45 min, 1.2 mM Mg²⁺, 0.64 mM dNTPs, 0.25 mM betaine, and 16:1 ratio of internal/external primers. The results showed that the established LAMP assay was able to specifically detect V. harveyi with a sensitivity of 3.4 × 10^–5 ng/µL. Clinical applicability analysis revealed that only V. harveyi was detected in the samples for pearl gentian grouper and whiteleg shrimp artificially infected by Vibrio. Therefore, we established a visual, reliable, rapid and sensitive LAMP-LFD method for detecting V. harveyi in aquatic animals.

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开发用于快速检测水生动物中 Harveyi 弧菌的可视环路介导等温扩增结合侧流浸量尺 (LAMP-LFD) 方法

Harveyi 弧菌是海水中的一种正常菌群，但近年来随着水产养殖的高密度化，Harveyi 弧菌已成为鱼虾的致病菌之一。本研究以 V. harveyi VieA 基因为靶标，设计引物并建立环介导等温扩增（LAMP）结合可视化侧流检测条（LFD）。优化的最佳反应条件为：60°C、45 min、1.2 mM Mg2+、0.64 mM dNTPs、0.25 mM 甜菜碱、内外引物比例为 16:1。结果表明，所建立的 LAMP 法能够特异性地检测 Harveyi 病毒，灵敏度为 3.4 × 10-5 ng/µL。临床适用性分析表明，在人工感染弧菌的珍珠龙胆石斑鱼和南美白对虾样本中，仅能检测到哈维氏弧菌。因此，我们建立了一种直观、可靠、快速和灵敏的 LAMP-LFD 方法来检测水生动物中的 Harveyi弧菌。

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来源期刊

Applied Biochemistry and Microbiology 生物-生物工程与应用微生物

CiteScore

1.70

自引率

12.50%

发文量

审稿时长

6-12 weeks

期刊介绍： Applied Biochemistry and Microbiology is an international peer reviewed journal that publishes original articles on biochemistry and microbiology that have or may have practical applications. The studies include: enzymes and mechanisms of enzymatic reactions, biosynthesis of low and high molecular physiologically active compounds; the studies of their structure and properties; biogenesis and pathways of their regulation; metabolism of producers of biologically active compounds, biocatalysis in organic synthesis, applied genetics of microorganisms, applied enzymology; protein and metabolic engineering, biochemical bases of phytoimmunity, applied aspects of biochemical and immunochemical analysis; biodegradation of xenobiotics; biosensors; biomedical research (without clinical studies). Along with experimental works, the journal publishes descriptions of novel research techniques and reviews on selected topics.