Development of a Taqman real-time PCR assay for rapid detection and quantification of Vibrio tapetis in extrapallial fluids of clams
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 se...
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
PeerJ Inc.
2015-12-01
|
| Series: | PeerJ |
| Subjects: | |
| Online Access: | https://peerj.com/articles/1484.pdf |
| _version_ | 1797422155004116992 |
|---|---|
| author | Adeline Bidault Gaëlle G. Richard Cédric Le Bris Christine Paillard |
| author_facet | Adeline Bidault Gaëlle G. Richard Cédric Le Bris Christine Paillard |
| author_sort | Adeline Bidault |
| collection | DOAJ |
| description | 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 CECT4600T 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 1.125 101 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. |
| first_indexed | 2024-03-09T07:28:06Z |
| format | Article |
| id | doaj.art-47ef32dd0ebd40e79cd4878f584e5d3e |
| institution | Directory Open Access Journal |
| issn | 2167-8359 |
| language | English |
| last_indexed | 2024-03-09T07:28:06Z |
| publishDate | 2015-12-01 |
| publisher | PeerJ Inc. |
| record_format | Article |
| series | PeerJ |
| spelling | doaj.art-47ef32dd0ebd40e79cd4878f584e5d3e2023-12-03T06:49:23ZengPeerJ Inc.PeerJ2167-83592015-12-013e148410.7717/peerj.1484Development of a Taqman real-time PCR assay for rapid detection and quantification of Vibrio tapetis in extrapallial fluids of clamsAdeline Bidault0Gaëlle G. Richard1Cédric Le Bris2Christine Paillard3Laboratoire des Sciences de l’Environnement Marin (LEMAR), UMR 6539 UBO/CNRS/IRD/Ifremer, Université de Bretagne Occidentale, Plouzané, FranceLaboratoire des Sciences de l’Environnement Marin (LEMAR), UMR 6539 UBO/CNRS/IRD/Ifremer, Université de Bretagne Occidentale, Plouzané, FranceLaboratoire des Sciences de l’Environnement Marin (LEMAR), UMR 6539 UBO/CNRS/IRD/Ifremer, Université de Bretagne Occidentale, Plouzané, FranceLaboratoire des Sciences de l’Environnement Marin (LEMAR), UMR 6539 UBO/CNRS/IRD/Ifremer, Université de Bretagne Occidentale, Plouzané, FranceThe 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 CECT4600T 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 1.125 101 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.https://peerj.com/articles/1484.pdfVibrio tapetisvirB4 geneTaqman real-time PCRMolecular diagnosticVenerupis philippinarumMarine pathogen |
| spellingShingle | Adeline Bidault Gaëlle G. Richard Cédric Le Bris Christine Paillard Development of a Taqman real-time PCR assay for rapid detection and quantification of Vibrio tapetis in extrapallial fluids of clams PeerJ Vibrio tapetis virB4 gene Taqman real-time PCR Molecular diagnostic Venerupis philippinarum Marine pathogen |
| title | Development of a Taqman real-time PCR assay for rapid detection and quantification of Vibrio tapetis in extrapallial fluids of clams |
| title_full | Development of a Taqman real-time PCR assay for rapid detection and quantification of Vibrio tapetis in extrapallial fluids of clams |
| title_fullStr | Development of a Taqman real-time PCR assay for rapid detection and quantification of Vibrio tapetis in extrapallial fluids of clams |
| title_full_unstemmed | Development of a Taqman real-time PCR assay for rapid detection and quantification of Vibrio tapetis in extrapallial fluids of clams |
| title_short | Development of a Taqman real-time PCR assay for rapid detection and quantification of Vibrio tapetis in extrapallial fluids of clams |
| title_sort | development of a taqman real time pcr assay for rapid detection and quantification of vibrio tapetis in extrapallial fluids of clams |
| topic | Vibrio tapetis virB4 gene Taqman real-time PCR Molecular diagnostic Venerupis philippinarum Marine pathogen |
| url | https://peerj.com/articles/1484.pdf |
| work_keys_str_mv | AT adelinebidault developmentofataqmanrealtimepcrassayforrapiddetectionandquantificationofvibriotapetisinextrapallialfluidsofclams AT gaellegrichard developmentofataqmanrealtimepcrassayforrapiddetectionandquantificationofvibriotapetisinextrapallialfluidsofclams AT cedriclebris developmentofataqmanrealtimepcrassayforrapiddetectionandquantificationofvibriotapetisinextrapallialfluidsofclams AT christinepaillard developmentofataqmanrealtimepcrassayforrapiddetectionandquantificationofvibriotapetisinextrapallialfluidsofclams |