Evaluation of a loop-mediated isothermal amplification method for the rapid detection of Vibrio harveyi in cultured marine shellfish

Abstract

The purpose of this study was to establish a loop-mediated isothermal amplification (LAMP) method for the rapid, sensitive detection of Vibrio harveyi in mariculture shellfish. A set of four primers, two outer and two inner primers, were designed from the toxR gene sequence of V. harveyi. The LAMP reaction was conducted at 65 degrees C for 60 min. There were no cross-reactions with other bacterial strains indicating a high specificity of the LAMP. The detection sensitivity of the LAMP assay for V. harveyi with both of pure cultures and added shellfish cultures is about 10(-5) dilution level (equivalent to 17.2 cells per reaction). The amplification products were detected by visual inspection using SYBR Green I. The detection sensitivity using the LAMP method was 10 times higher than that of conventional PCR. The LAMP assay established in this study is an extremely specific, sensitive and rapid for identification of V. harveyi in mariculture shellfish. This LAMP technique provides an important detecting tool for the detection of V. harveyi infection both in the laboratory and field. This technique is recommended as an applied protocol for health management programme and disease surveillance of in hatcheries as well as in grow-out pond, to prevent the disease outbreak.