gbpA as a Novel qPCR Target for the Species-Specific Detection of Vibrio cholerae O1, O139, Non-O1/Non-O139 in Environmental, Stool, and Historical Continuous Plankton Recorder Samples

dc.contributor.authorVezzulli, Luigien_ZA
dc.contributor.authorStauder, Monicaen_ZA
dc.contributor.authorGrande, Chiaraen_ZA
dc.contributor.authorPezzati, Elisabettaen_ZA
dc.contributor.authorVerheye, Hans M.en_ZA
dc.contributor.authorOwens, Nicholas J. P.en_ZA
dc.contributor.authorPruzzo, Carlaen_ZA
dc.date.accessioned2015-11-09T13:19:28Z
dc.date.available2015-11-09T13:19:28Z
dc.date.issued2015en_ZA
dc.description.abstractThe Vibrio cholerae N-acetyl glucosamine-binding protein A (GbpA) is a chitin-binding protein involved in V . cholerae attachment to environmental chitin surfaces and human intestinal cells. We previously investigated the distribution and genetic variations of gbpA in a large collection of V . cholerae strains and found that the gene is consistently present and highly conserved in this species. Primers and probe were designed from the gbpA sequence of V . cholerae and a new Taq-based qPCR protocol was developed for diagnostic detection and quantification of the bacterium in environmental and stool samples. In addition, the positions of primers targeting the gbpA gene region were selected to obtain a short amplified fragment of 206 bp and the protocol was optimized for the analysis of formalin-fixed samples, such as historical Continuous Plankton Recorder (CPR) samples. Overall, the method is sensitive (50 gene copies), highly specific for V . cholerae and failed to amplify strains of the closely-related species Vibrio mimicus . The sensitivity of the assay applied to environmental and stool samples spiked with V . cholerae ATCC 39315 was comparable to that of pure cultures and was of 10 2 genomic units/l for drinking and seawater samples, 10 1 genomic units/g for sediment and 10 2 genomic units/g for bivalve and stool samples. The method also performs well when tested on artificially formalin-fixed and degraded genomic samples and was able to amplify V . cholerae DNA in historical CPR samples, the earliest of which date back to August 1966. The detection of V . cholerae in CPR samples collected in cholera endemic areas such as the Benguela Current Large Marine Ecosystem (BCLME) is of particular significance and represents a proof of concept for the possible use of the CPR technology and the developed qPCR assay in cholera studies.en_ZA
dc.identifier.apacitationVezzulli, L., Stauder, M., Grande, C., Pezzati, E., Verheye, Hans M., Owens, Nicholas J. P., & Pruzzo, C. (2015). gbpA as a Novel qPCR Target for the Species-Specific Detection of Vibrio cholerae O1, O139, Non-O1/Non-O139 in Environmental, Stool, and Historical Continuous Plankton Recorder Samples. <i>PLoS One</i>, http://hdl.handle.net/11427/14786en_ZA
dc.identifier.chicagocitationVezzulli, Luigi, Monica Stauder, Chiara Grande, Elisabetta Pezzati, Hans M. Verheye, Nicholas J. P. Owens, and Carla Pruzzo "gbpA as a Novel qPCR Target for the Species-Specific Detection of Vibrio cholerae O1, O139, Non-O1/Non-O139 in Environmental, Stool, and Historical Continuous Plankton Recorder Samples." <i>PLoS One</i> (2015) http://hdl.handle.net/11427/14786en_ZA
dc.identifier.citationVezzulli, L., Stauder, M., Grande, C., Pezzati, E., Verheye, H. M., Owens, N. J., & Pruzzo, C. (2015). gbpA as a Novel qPCR Target for the Species-Specific Detection of Vibrio cholerae O1, O139, Non-O1/Non-O139 in Environmental, Stool, and Historical Continuous Plankton Recorder Samples. doi:10.1371/journal.pone.0123983en_ZA
dc.identifier.ris TY - Journal Article AU - Vezzulli, Luigi AU - Stauder, Monica AU - Grande, Chiara AU - Pezzati, Elisabetta AU - Verheye, Hans M. AU - Owens, Nicholas J. P. AU - Pruzzo, Carla AB - The Vibrio cholerae N-acetyl glucosamine-binding protein A (GbpA) is a chitin-binding protein involved in V . cholerae attachment to environmental chitin surfaces and human intestinal cells. We previously investigated the distribution and genetic variations of gbpA in a large collection of V . cholerae strains and found that the gene is consistently present and highly conserved in this species. Primers and probe were designed from the gbpA sequence of V . cholerae and a new Taq-based qPCR protocol was developed for diagnostic detection and quantification of the bacterium in environmental and stool samples. In addition, the positions of primers targeting the gbpA gene region were selected to obtain a short amplified fragment of 206 bp and the protocol was optimized for the analysis of formalin-fixed samples, such as historical Continuous Plankton Recorder (CPR) samples. Overall, the method is sensitive (50 gene copies), highly specific for V . cholerae and failed to amplify strains of the closely-related species Vibrio mimicus . The sensitivity of the assay applied to environmental and stool samples spiked with V . cholerae ATCC 39315 was comparable to that of pure cultures and was of 10 2 genomic units/l for drinking and seawater samples, 10 1 genomic units/g for sediment and 10 2 genomic units/g for bivalve and stool samples. The method also performs well when tested on artificially formalin-fixed and degraded genomic samples and was able to amplify V . cholerae DNA in historical CPR samples, the earliest of which date back to August 1966. The detection of V . cholerae in CPR samples collected in cholera endemic areas such as the Benguela Current Large Marine Ecosystem (BCLME) is of particular significance and represents a proof of concept for the possible use of the CPR technology and the developed qPCR assay in cholera studies. DA - 2015 DB - OpenUCT DO - 10.1371/journal.pone.0123983 DP - University of Cape Town J1 - PLoS One LK - https://open.uct.ac.za PB - University of Cape Town PY - 2015 T1 - gbpA as a Novel qPCR Target for the Species-Specific Detection of Vibrio cholerae O1, O139, Non-O1/Non-O139 in Environmental, Stool, and Historical Continuous Plankton Recorder Samples TI - gbpA as a Novel qPCR Target for the Species-Specific Detection of Vibrio cholerae O1, O139, Non-O1/Non-O139 in Environmental, Stool, and Historical Continuous Plankton Recorder Samples UR - http://hdl.handle.net/11427/14786 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/14786
dc.identifier.urihttp://dx.doi.org/10.1371/journal.pone.0123983
dc.identifier.vancouvercitationVezzulli L, Stauder M, Grande C, Pezzati E, Verheye Hans M, Owens Nicholas J P, et al. gbpA as a Novel qPCR Target for the Species-Specific Detection of Vibrio cholerae O1, O139, Non-O1/Non-O139 in Environmental, Stool, and Historical Continuous Plankton Recorder Samples. PLoS One. 2015; http://hdl.handle.net/11427/14786.en_ZA
dc.language.isoengen_ZA
dc.publisherPublic Library of Scienceen_ZA
dc.publisher.departmentMarine Research (MA-RE) Instituteen_ZA
dc.publisher.facultyFaculty of Scienceen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.rightsThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.en_ZA
dc.rights.holder© 2015 Vezzulli et alen_ZA
dc.rights.urihttp://creativecommons.org/licenses/by/4.0en_ZA
dc.sourcePLoS Oneen_ZA
dc.source.urihttp://journals.plos.org/plosoneen_ZA
dc.subject.otherVibrio choleraeen_ZA
dc.subject.otherPolymerase chain reactionen_ZA
dc.subject.otherCholeraen_ZA
dc.subject.otherDNA extractionen_ZA
dc.subject.otherPlanktonen_ZA
dc.subject.otherSedimenten_ZA
dc.subject.otherSea wateren_ZA
dc.subject.otherGene amplificationen_ZA
dc.titlegbpA as a Novel qPCR Target for the Species-Specific Detection of Vibrio cholerae O1, O139, Non-O1/Non-O139 in Environmental, Stool, and Historical Continuous Plankton Recorder Samplesen_ZA
dc.typeJournal Articleen_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceArticleen_ZA
Files
Original bundle
Now showing 1 - 1 of 1
Loading...
Thumbnail Image
Name:
Vezzulli_gbpA_as_a_Novel_qPCR_Target_2015.pdf
Size:
712.98 KB
Format:
Adobe Portable Document Format
Description:
Collections