An improved method for detection of Edwardsiella tarda by loop-mediated isothermal amplification by targeting the EsrB gene |
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| Authors: | Guosi Xie Qingli Zhang Nana Han Chengyin Shi Xiuhua Wang Qinghui Liu Jie Huang |
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| Institution: | 1. Key Laboratory of Marine Fishery Resources Sustainable Utilization, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, 266071, China
2. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai, 201306, China
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| Abstract: | Edwardsiella tarda is a major pathogen in aquatic environments that can cause heavy economic losses. An improved method for quick and accurate detection of E. tarda by loop-mediated isothermal amplification (LAMP) with two additional loop primers was developed by targeting the EsrB gene (EsrB — LAMP). In this method, the Mg2+ concentration, reaction temperature, and reaction time were optimized to 8 mmol/L, 61°C, and 40 min, respectively. The detection limit with the EsrB gene was as low as 10 copies, which is 100 times more sensitive than that of conventional polymerase chain reaction (PCR). The EsrB-LAMP assay was shown more sensitive and rapid than previously reported LAMP assays targeting the hemolysin gene ( hemolysin -LAMP) for detection of E. tarda. The EsrB -LAMP was also highly specific to E. tarda and had no cross-reaction with 13 other strains of bacteria. The assay can be carried out in a simple heating device and the EsrB-LAMP products can be visually detected by adding fluorescent dye to the reaction mixture. Taken together, the improved EsrB-LAMP diagnostic protocol has the potential for detection of E. tarda from indoor and outdoor samples. |
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| Keywords: | Edwardsiella tarda LAMP detection EsrB |
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