Detection of Pathogenic Vibrio anguillarum by Using Duplex PCR and LAMP Assays

Abstract:

Vibrio anguillarum widely exists in aquatic environments and has been recognized as one of the prominent waterborne pathogens that undermine the aquaculture industry worldwide. In this study, we investigated the prevalent distribution of eight virulence-associated genes in the V. anguillarum strains isolated from Scophthalmus maximus, Cynoglossus semilaevis and Cyprinus carpio, and improved the detection of V. anguillarum by using duplex PCR and LAMP assays. Six genes (empA, vah1, vah4, flaA, rtxA, and tonB) were detected in all 22 pathogenic strains of V. anguillarum, but virA and angM were not detected. The duplex PCR assay was established with vah4 and rtxA genes as molecular markers. Two gene fragments from the chromosomal DNA of V. anguillarum were detected in one PCR reaction with the detection limit of 2.4×103 CFU/ml. The assay in 6 other control strains generated negative results. The LAMP assay was established with vah4 as the molecular marker. The positive reaction was shown as stair-step amplified bands and the detection limit was 2.4×101 CFU/ml. The assay produced negative reactions in 6 types of control pathogenic bacteria (no amplified bands). The LAMP method was 100 times more sensitive than the PCR method. Therefore we concluded that the LAMP assay could be a sensitive, rapid and simple tool for the detection of V. anguillarum, and recommend to employ this method in the early diagnosis of V. anguillarum infection in aquatic animals.