Molecular identification of Vibrio tapetis, the causative agent of the brown ring disease of Ruditapes philippinarum

Vibrio tapetis is the marine bacterium responsible for the brown ring disease (BRD) affecting the manila clam, Ruditapes philippinarum. Identification of V. tapetis has been previously performed using biochemical criteria and serological procedures. All of these methods are time consuming and ill-adapted to individual screening. This study describes an oligonucleotidic probe (Vt446) and two PCR primers, deduced from the 16S rDNA sequence, allowing a fast and specific V. tapetis identification using dot blot hybridisation and species-specific primed PCR (SSP-PCR). The probe and primers have been tested on 60 strains, including referenced Vibrio sp., Gram negative and positive bacteria, marine bacteria samples and isolated clam bacteria. For all the 19 V. tapetis strains, the results of PCR assays consistently corroborated those of the agglutination tests. The detection limit was estimated to be 102 CFU ml− 1. The SSP-PCR method has resulted in V. tapetis detection in larvae, in diseased clams, and in asymptomatic broodstock clams that later developed BRD. In conclusion, the two SSP-PCR primers were useful for direct and fast identification of V. tapetis strains isolated in clams, and are well suited for the screening of individual R. philippinarum broodstock clams and larvae from the hatchery.