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Visual closed-tube loop-mediated isothermal amplification (LAMP) assay targeting aerolysin gene: a practical screening method for virulent Aeromonas species affecting cultured eels in China

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Abstract

Under intensive and high-density aquaculture conditions, the cultured eels are easily infected by a variety of virulent Aeromonas species, which leads to high mortality rates and economic losses. In this study, a visual closed-tube loop-mediated isothermal amplification (LAMP) assay was developed for targeting aerolysin gene of Aeromonas species affecting cultured eels. Firstly, two pairs of outer and inner primers were designed against a conserved region of the aer gene from different Aeromonas species. Secondly, the weakly buffered aer-LAMP assay was optimized under various Mg2+ concentrations, different amplification times and amplification temperatures followed by the visual evaluation of phenol red dye and SYTO-9. Thirdly, the detection limit of the aer-LAMP assay showed 16.8 copy/μl which was simultaneously evaluated by the color changes from red to yellow and the real-time fluorescence amplification curves. The specificity of the aer-LAMP assay showed no cross-reaction with other related species. In addition, 30 Aeromonas strains and 40 clinical tissues from eels were used to further validate the effectiveness of the aer-LAMP assay. All the results showed that the aer-LAMP assay provided a rapid, simple, and reliable method for detecting Aeromonas harboring the aerolysin gene, which would be helpful for the prevention and control of Aeromonas infection in cultured eels.

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Funding

This work was supported by the Open Project of Key Laboratory of Aquaculture Disease Control, Ministry of Agriculture; Department of Education, Fujian Province [JA170301]; Natural Science Foundation of Fujian Province [2018J01452]; Jimei University National Natural Science Foundation Training Project [ZP2020025]; and Special Fund for the Adjustment of Fishery Structure in Fujian Province [2020].

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Authors and Affiliations

  1. Key Laboratory of Aquaculture Disease Control, Ministry of Agriculture, Wuhan, People’s Republic of China

    Jing Xiong, Shan-gong Wu & Yi-bing Zhang

  2. Fisheries College, Jimei University, Xiamen, People’s Republic of China

    Jing Xiong, Ying Liang, Yue-lian Zou, Xiao-mei Xie & Wen-shu Huang

  3. Engineering Research Center of the Modern Technology for Eel Industry, Ministry of Education, Xiamen, People’s Republic of China

    Jing Xiong, Ying Liang & Wen-shu Huang

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  1. Jing Xiong
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  2. Shan-gong Wu
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  3. Ying Liang
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  4. Yue-lian Zou
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  5. Xiao-mei Xie
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  6. Wen-shu Huang
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  7. Yi-bing Zhang
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Correspondence to Wen-shu Huang.

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We ensured that the experiments followed the ethical guidelines of Jimei University and and confirmed that all experimental protocols were approved by Jimei University.

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Xiong, J., Wu, Sg., Liang, Y. et al. Visual closed-tube loop-mediated isothermal amplification (LAMP) assay targeting aerolysin gene: a practical screening method for virulent Aeromonas species affecting cultured eels in China. Aquacult Int 28, 2319–2332 (2020). https://doi.org/10.1007/s10499-020-00588-z

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  • DOI: https://doi.org/10.1007/s10499-020-00588-z

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