Development of a Taqman real-time PCR assay for rapid detection and quantification ofVibrio tapetisin extrapallial fluids of clams

Abstract

The Gram-negative bacteriumVibrio tapetisis known as the causative agent of Brown Ring Disease (BRD) in the Manila clamVenerupis(=Ruditapes)philippinarum. This bivalve is the second most important species produced in aquaculture and has a high commercial value. In spite of the development of several molecular methods, no survey has been yet achieved to rapidly quantify the bacterium in the clam. In this study, we developed a Taqman real-time PCR assay targeting virB4 gene for accurate and quantitative identification ofV. tapetisstrains pathogenic to clams. Sensitivity and reproducibility of the method were assessed using either filtered sea water or extrapallial fluids of clam injected with the CECT4600^TV. tapetisstrain. Quantification curves ofV. tapetisstrain seeded in filtered seawater (FSW) or extrapallial fluids (EF) samples were equivalent showing reliable qPCR efficacies. With this protocol, we were able to specifically detectV. tapetisstrains down to 1.125 10¹bacteria per mL of EF or FSW, taking into account the dilution factor used for appropriate template DNA preparation. This qPCR assay allowed us to monitorV. tapetisload both experimentally or naturally infected Manila clams. This technique will be particularly useful for monitoring the kinetics of massive infections byV. tapetisand for designing appropriate control measures for aquaculture purposes.