Development of a loop-mediated isothermal amplification method for detection of Shewanella algae in fish

Abstract

Shewanella algae is an opportunistic pathogen, which is widely distributed in freshwater and marine environments. Diseases caused by S. algae have resulted in substantial economic losses in aquaculture industry. This study aimed to establish a loop mediated isothermal amplification (LAMP) method for rapid and specific detection of S. algae in aquatic animals. The specific LAMP primers were designed according to the 16 S rRNA gene sequences of S. algae strains deposited in GenBank. Based on adjustment of the primer concentration and optimization of reaction conditions, the LAMP method for S. algae detection was established, and specificity and sensitivity tests were carried out. The optimal temperature of this method was 65 °C at constant 1 h. The LAMP amplification reaction was positive for S. algae, but negative for Pseudoalteromonas pisicida, Aeromonas hydrophila, A. veronii, Vibrio parahemolyticus, S. decolorationis, S. xiamenensis, Enterobacter cloacae, Bacillus cereus and Staphylococcus epidermidis. The minimum concentration of template DNA of S. algae for the LAMP method was 8.15 × 10−5 µg/µL. The LAMP method was comparable to the common PCR when they were used for detection of S. algae in koi carp artificially infected with this pathogen. In this study, a rapid, specific and sensitive LAMP method was successfully established for the detection of S. algae, providing great convenience for rapid diagnosis of aquatic animal diseases caused by S. algae infection.