Development of a sensitive polymerase chain reaction (PCR) and digoxigenin (DIG)-labeled in situ hybridization (ISH) for the detection of Vibrio bacteria in the Pacific oyster Crassostrea gigas

Vibrio bacteria are one of the greatest threats to aquaculture, with Vibrio-associated mass mortality events occurring in all life stages worldwide. Vibrio aestuarianus is particularly problematic in Pacific oyster Crassostrea gigas cultures. A diagnostic method used to detect V. aestuarianus is qua...

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Autores principales:	Gary F. Kett, Sarah C. Culloty, Marcel A.K. Jansen, Sharon A. Lynch
Formato:	article
Lenguaje:	EN
Publicado:	Elsevier 2022
Materias:	Aquaculture Bacteria Diagnostics In situ hybridization PCR Vibrio Aquaculture. Fisheries. Angling SH1-691
Acceso en línea:	https://doaj.org/article/5069ecd3bb6b45db8560165c3f93d987
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spelling	oai:doaj.org-article:5069ecd3bb6b45db8560165c3f93d9872021-12-02T05:01:58ZDevelopment of a sensitive polymerase chain reaction (PCR) and digoxigenin (DIG)-labeled in situ hybridization (ISH) for the detection of Vibrio bacteria in the Pacific oyster Crassostrea gigas2352-513410.1016/j.aqrep.2021.100961https://doaj.org/article/5069ecd3bb6b45db8560165c3f93d9872022-02-01T00:00:00Zhttp://www.sciencedirect.com/science/article/pii/S2352513421003781https://doaj.org/toc/2352-5134Vibrio bacteria are one of the greatest threats to aquaculture, with Vibrio-associated mass mortality events occurring in all life stages worldwide. Vibrio aestuarianus is particularly problematic in Pacific oyster Crassostrea gigas cultures. A diagnostic method used to detect V. aestuarianus is quantitative (q)PCR and a confirmatory method is fluorescent in situ hybridization (FISH). These methods require specialized equipment and incur high costs. The objective of this study was to develop a novel generic (relating to a genus) PCR and DIG-labeled in situ hybridization (ISH) for the detection and confirmation of Vibrio. A PCR that detects all Vibrio spp. is particularly useful for blanket screening of hosts and environmental samples, while ISH facilitates the visual localization of bacteria. Both diagnostic methods were developed to be used synchronously with other methods that are specific to certain Vibrio spp. Primers (VibF3/VibR3) were designed to amplify a 286 bp product from the 16S ribosomal RNA gene common to all Vibrio spp. and to form the ISH probe. Confirmatory positive and negative controls were used in both methods. Results were compared with a V. aestuarianus specific qPCR protocol. Direct Sanger sequencing of PCR products, Blastn analysis and Clustal Omega alignments were used to confirm Vibrio sp. detection and assess similarity. Pathologies observed in C. gigas tissues were similar to those described in other studies. A positive ISH signal was observed in qPCR and PCR positive oysters and no signal was detected in uninfected individuals. These techniques will increase the diagnostic arsenal against this globally significant genus of bacteria.Gary F. KettSarah C. CullotyMarcel A.K. JansenSharon A. LynchElsevierarticleAquacultureBacteriaDiagnosticsIn situ hybridizationPCRVibrioAquaculture. Fisheries. AnglingSH1-691ENAquaculture Reports, Vol 22, Iss , Pp 100961- (2022)
institution	DOAJ
collection	DOAJ
language	EN
topic	Aquaculture Bacteria Diagnostics In situ hybridization PCR Vibrio Aquaculture. Fisheries. Angling SH1-691
spellingShingle	Aquaculture Bacteria Diagnostics In situ hybridization PCR Vibrio Aquaculture. Fisheries. Angling SH1-691 Gary F. Kett Sarah C. Culloty Marcel A.K. Jansen Sharon A. Lynch Development of a sensitive polymerase chain reaction (PCR) and digoxigenin (DIG)-labeled in situ hybridization (ISH) for the detection of Vibrio bacteria in the Pacific oyster Crassostrea gigas
description	Vibrio bacteria are one of the greatest threats to aquaculture, with Vibrio-associated mass mortality events occurring in all life stages worldwide. Vibrio aestuarianus is particularly problematic in Pacific oyster Crassostrea gigas cultures. A diagnostic method used to detect V. aestuarianus is quantitative (q)PCR and a confirmatory method is fluorescent in situ hybridization (FISH). These methods require specialized equipment and incur high costs. The objective of this study was to develop a novel generic (relating to a genus) PCR and DIG-labeled in situ hybridization (ISH) for the detection and confirmation of Vibrio. A PCR that detects all Vibrio spp. is particularly useful for blanket screening of hosts and environmental samples, while ISH facilitates the visual localization of bacteria. Both diagnostic methods were developed to be used synchronously with other methods that are specific to certain Vibrio spp. Primers (VibF3/VibR3) were designed to amplify a 286 bp product from the 16S ribosomal RNA gene common to all Vibrio spp. and to form the ISH probe. Confirmatory positive and negative controls were used in both methods. Results were compared with a V. aestuarianus specific qPCR protocol. Direct Sanger sequencing of PCR products, Blastn analysis and Clustal Omega alignments were used to confirm Vibrio sp. detection and assess similarity. Pathologies observed in C. gigas tissues were similar to those described in other studies. A positive ISH signal was observed in qPCR and PCR positive oysters and no signal was detected in uninfected individuals. These techniques will increase the diagnostic arsenal against this globally significant genus of bacteria.
format	article
author	Gary F. Kett Sarah C. Culloty Marcel A.K. Jansen Sharon A. Lynch
author_facet	Gary F. Kett Sarah C. Culloty Marcel A.K. Jansen Sharon A. Lynch
author_sort	Gary F. Kett
title	Development of a sensitive polymerase chain reaction (PCR) and digoxigenin (DIG)-labeled in situ hybridization (ISH) for the detection of Vibrio bacteria in the Pacific oyster Crassostrea gigas
title_short	Development of a sensitive polymerase chain reaction (PCR) and digoxigenin (DIG)-labeled in situ hybridization (ISH) for the detection of Vibrio bacteria in the Pacific oyster Crassostrea gigas
title_full	Development of a sensitive polymerase chain reaction (PCR) and digoxigenin (DIG)-labeled in situ hybridization (ISH) for the detection of Vibrio bacteria in the Pacific oyster Crassostrea gigas
title_fullStr	Development of a sensitive polymerase chain reaction (PCR) and digoxigenin (DIG)-labeled in situ hybridization (ISH) for the detection of Vibrio bacteria in the Pacific oyster Crassostrea gigas
title_full_unstemmed	Development of a sensitive polymerase chain reaction (PCR) and digoxigenin (DIG)-labeled in situ hybridization (ISH) for the detection of Vibrio bacteria in the Pacific oyster Crassostrea gigas
title_sort	development of a sensitive polymerase chain reaction (pcr) and digoxigenin (dig)-labeled in situ hybridization (ish) for the detection of vibrio bacteria in the pacific oyster crassostrea gigas
publisher	Elsevier
publishDate	2022
url	https://doaj.org/article/5069ecd3bb6b45db8560165c3f93d987
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Development of a sensitive polymerase chain reaction (PCR) and digoxigenin (DIG)-labeled in situ hybridization (ISH) for the detection of Vibrio bacteria in the Pacific oyster Crassostrea gigas

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