1. 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.
- Author
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Xiong, Jing, Wu, Shan-gong, Liang, Ying, Zou, Yue-lian, Xie, Xiao-mei, Huang, Wen-shu, and Zhang, Yi-bing
- Subjects
AEROMONAS ,GENE targeting ,EELS ,INFECTION control ,MALACHITE green ,NUCLEIC acid amplification techniques ,GENE amplification - 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 Mg
2+ 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. [ABSTRACT FROM AUTHOR]- Published
- 2020
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