Specific and Rapid Diagnosis of Edwardsiella tarda by a Novel Loop-Mediated Isothermal Amplification Targeting the Upstream Region of hlyb Gene

Abstract

Edwardsiella tarda has become one of the most severe pathogens in aquaculture industries throughout the world; therefore, a specific and rapid identification method for this bacterium is urgently needed. In the present study, a novel loop-mediated isothermal amplification (LAMP) was developed by targeting the upstream region of the hlyb gene of E. tarda, which was then named as UH-LAMP. The Mg²⁺ concentrations, the reaction temperature, and the reaction time of UH-LAMP were optimized to 10 mM, 65°C, and 45 min, respectively. The detection limit of the UH-LAMP was 100-times higher than that of conventional polymerase chain reaction (10 versus 1000 CFU/test). Furthermore, the new UH-LAMP assay showed no cross-reactivity to the E. ictaluri belonging to the other species in the genus Edwardsiella. The high specificity of the assay was also confirmed by testing the nine strains of E. tarda collected from different geographical locations and the other 20 bacteria species. The assay can be performed in a simple water bath or a heat block and the detection result can be visualized by adding a fluorescent reagent to the reaction mixture. Taken together, our preliminary results indicate that this UH-LAMP assay provided a rapid, sensitive, and species-specific diagnostic tool for E. tarda and can easily be applied for the diagnosis under clinical or onsite conditions.

Received December 10, 2012; accepted February 22, 2013

ACKNOWLEDGMENTS

This work was supported by the Special Fund for Agro-scientific Research in the Public Interest (No. 201103034), the National Key Technology R&D Program (Grant No. 2012BAD17B01), and the Construction Special Fund of Modern Agriculture and Industrial Technology Research System (No. CARS-47). We thank Professor Cheng-Ping Lu at the Nanjing Agricultural University for providing us with the E. tarda strain CD and Dr. Ai-Hua Li at the Chinese Academy of Sciences for providing us with the E. ictaluri strains HSN-1, Dr. Zhao-Lan Mo at the Chinese Academy of Sciences for providing us with the Aeromonas hydrophila strain LSA34, and Professor Chang-Fu Cheng at the Huazhong Agricultural University for providing us with the A. hydrophila strain 052901.