title: Development of a PCR-free DNA-based assay for the specific detection of Vibrio species in environmental samples by targeting the 16S rRNA.
abstract: A novel PCR-free DNA-based assay was developed for the detection of Vibrio spp. A sandwich hybridization format using an immobilized capture probe and a labeled signal probe was selected and combined with chemiluminescent method for the detection of the RNA target. In a first step, probes were validated using positive controls (PCs). A linearity was observed between 0.1 and 2.5 nM of PC, and detection limit was determined as 0.1 nM. In a second step, specificity was checked by using RNA extracted from a panel of 31 environmental bacterial strains. Detection limit of 5 ng μL-1of total fragmented RNA was obtained, and the assay allowed a good discrimination between the 21 Vibrio and the 10 non-Vibrio strains tested. Finally, the DNA-based assay was successfully applied to analysis of spiked and natural environmental samples. Stability and analysis time of the DNA-based assay were also investigated to optimize working conditions. We demonstrated that microplates can be coated beforehand with capture probe and stored at 4 °C without any buffer in wells for at least 30 days. The use of the pre-made plates enables the assay to be completed in 2 h. The developed assay appeared as an interesting tool to determine the presence of bacteria in environmental samples.
authors.affiliation: Biocapteurs Analyses Environment, Université Perpignan, Via Domitia, 66860, Perpignan, France.|CNRS, Laboratoire de Biodiversité et Biotechnologies Microbiennes (LBBM), Observatoire Océanologique, Sorbonne Universités, UPMC Univ. Paris 06, F-66650, Banyuls/Mer, France.
authors.forename: E
authors.initials: E
authors.lastname: Da-Silva
authors.affiliation: Biocapteurs Analyses Environment, Université Perpignan, Via Domitia, 66860, Perpignan, France. barthelm@univ-perp.fr.
authors.forename: L
authors.identifier: http://orcid.org/0000-0002-1961-0701
authors.initials: L
authors.lastname: Barthelmebs
authors.affiliation: CNRS, Laboratoire de Biodiversité et Biotechnologies Microbiennes (LBBM), Observatoire Océanologique, Sorbonne Universités, UPMC Univ. Paris 06, F-66650, Banyuls/Mer, France.
authors.forename: J
authors.initials: J
authors.lastname: Baudart
mesh_terms: D004269:DNA, Bacterial / Q000235:genetics; D004279:DNA, Viral; D004784:Environmental Monitoring / Q000379:methods*; D057230:Limit of Detection; D009693:Nucleic Acid Hybridization / Q000379:methods; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D014733:Vibrio / Q000235:genetics*
chemical_list: D004269:DNA, Bacterial; D004279:DNA, Viral; D012336:RNA, Ribosomal, 16S
keywords: Chemiluminescence; DNA-based assay; Environmental analysis; PCR-free; Sandwich hybridization assay; Vibrio detection