Screening of vibriosis in Asian seabass, Lates calcarifer using loop-mediated isothermal amplification (LAMP) assay

Abstract

The aim of this study was to standardize a loop-mediated isothermal amplification (LAMP) assay for the detection of <i>Vibrio harveyi</i> , the causative agent of vibriosis in Asian seabass, <i>Lates calcarifer</i>. The dnaJ gene of the bacterial pathogen was used as the target gene for the LAMP assay. It was optimized at an incubation time of 1 h at 63°C. The assay was highly specific for <i>V. harveyi</i> and did not cross-react with other bacterial pathogens offish. However, the assay was able to detect <i>V. harveyi</i> that was isolated from infected shrimps. The limit of detection of the LAMP assay was 40 pg of DNA mL^-1 or 40 fg of the genomic DNA per LAMP reaction and was 10 times more sensitive than conventional PCR in detecting the bacterial pathogen from infected samples. The LAMP products can be quantified spectrophotometrically using hydroxynaphthol blue (HNB) dye and showed positive correlation with the amount of the pathogen. These results demonstrated that LAMP is a simple and sensitive detection technique that has potential application for routine diagnosis of vibrosis caused by <i>V. harveyi</i> in Asian seabass and other aquatic species.