title: Development of a Taqman real-time PCR assay for rapid detection and quantification of Vibrio tapetis in extrapallial fluids of clams.
abstract: The Gram-negative bacterium Vibrio tapetis is known as the causative agent of Brown Ring Disease (BRD) in the Manila clam Venerupis (=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 of V. tapetis strains 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(T) V. tapetis strain. Quantification curves of V. tapetis strain seeded in filtered seawater (FSW) or extrapallial fluids (EF) samples were equivalent showing reliable qPCR efficacies. With this protocol, we were able to specifically detect V. tapetis strains down to 1.125 10(1) bacteria per mL of EF or FSW, taking into account the dilution factor used for appropriate template DNA preparation. This qPCR assay allowed us to monitor V. tapetis load both experimentally or naturally infected Manila clams. This technique will be particularly useful for monitoring the kinetics of massive infections by V. tapetis and for designing appropriate control measures for aquaculture purposes.
authors.affiliation: Laboratoire des Sciences de l'Environnement Marin (LEMAR), UMR 6539 UBO/CNRS/IRD/Ifremer, Université de Bretagne Occidentale , Plouzané , France.
authors.forename: Adeline
authors.initials: A
authors.lastname: Bidault
authors.affiliation: Laboratoire des Sciences de l'Environnement Marin (LEMAR), UMR 6539 UBO/CNRS/IRD/Ifremer, Université de Bretagne Occidentale , Plouzané , France.
authors.forename: Gaëlle G
authors.initials: GG
authors.lastname: Richard
authors.affiliation: Laboratoire des Sciences de l'Environnement Marin (LEMAR), UMR 6539 UBO/CNRS/IRD/Ifremer, Université de Bretagne Occidentale , Plouzané , France.
authors.forename: Cédric
authors.initials: C
authors.lastname: Le Bris
authors.affiliation: Laboratoire des Sciences de l'Environnement Marin (LEMAR), UMR 6539 UBO/CNRS/IRD/Ifremer, Université de Bretagne Occidentale , Plouzané , France.
authors.forename: Christine
authors.initials: C
authors.lastname: Paillard
keywords: Brown ring disease; Marine pathogen; Molecular diagnostic; Taqman real-time PCR; Venerupis philippinarum; Vibrio tapetis; virB4 gene