Visual closed-tube loop-mediated isothermal amplification (LAMP) assay targeting aerolysin gene: a practical screening m
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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 Jing Xiong 1,2,3 & Shan-gong Wu 1 & Ying Liang 2,3 & Yue-lian Zou 2 & Xiao-mei Xie 2 & Wen-shu Huang 2,3 & Yi-bing Zhang 1 Received: 23 March 2020 / Accepted: 4 August 2020/ # Springer Nature Switzerland AG 2020
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. Keywords Aeromonas spp. . Closed-tube loop-mediated isothermal amplification (LAMP) assay . Aerolysin (aer) gene . Cultured eels
* Wen-shu Huang [email protected]
1
Key Laboratory of Aquaculture Disease Control, Ministry of Agriculture, Wuhan, People’s Republic of China
2
Fisheries College, Jimei University, Xiamen, People’s Republic of China
3
Engineering Research Center of the Modern Technology for Eel Industry, Ministry of Education, Xiamen, People’s Republic of China
Aquaculture International
Introduction Anguilla spp. (Anguilla japonica, A. anguilla, A. rostrate, A. marmorata, A. bicolor pacifica), commonly known as eels, have delicious meat and high nutritional value (Guan et al. 2011). As important economic fishes, eel production in China accounts for more than 70% of the worldwide production (Guo et al. 2016). However, under the intensive and high-density aquaculture conditions, the cultured eels are easily infected by a variety of pathogenic Aeromonas species, such as A. veronii, A. hydrophila, A.sobria, and A. caviae (Guo et al. 2016; Xiong et al. 2019). The pathogenicity of Aeromonas is considered to be related to a variety of virulence factors, such as aerolysin (encoded by the gene aer), hemolysin (hly), enterotox
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