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.
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 102 genomic units/l for drinking and seawater samples, 101 genomic units/g for sediment and 102 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.
Department of Earth, Environmental and Life Sciences (DISTAV), University of Genoa, Genoa, Italy.
Luigi
L
Vezzulli
Department of Earth, Environmental and Life Sciences (DISTAV), University of Genoa, Genoa, Italy.
Monica
M
Stauder
Department of Earth, Environmental and Life Sciences (DISTAV), University of Genoa, Genoa, Italy.
Chiara
C
Grande
Department of Earth, Environmental and Life Sciences (DISTAV), University of Genoa, Genoa, Italy.
Elisabetta
E
Pezzati
Oceans and Coastal Research, Department of Environmental Affairs (DEA) and Marine Research Institute, University of Cape Town (UCT), Cape Town, South Africa.
Hans M
HM
Verheye
Sir Alister Hardy Foundation for Ocean Science (SAHFOS), Plymouth, United Kingdom.
Nicholas J P
NJ
Owens
Department of Earth, Environmental and Life Sciences (DISTAV), University of Genoa, Genoa, Italy.
Carla
C
Pruzzo
D005243:Feces / Q000382:microbiology*; D005798:Genes, Bacterial; D006801:Humans; D064307:Microbiota / Q000235:genetics*; D058889:Molecular Typing / Q000379:methods*; D010933:Plankton / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods; D012623:Seawater / Q000382:microbiology*; D012680:Sensitivity and Specificity; D014734:Vibrio cholerae / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification