Development of a PCR-free DNA-based assay for the specific detection of Vibrio species in environmental samples by targeting the 16S rRNA

• Published in: Environmental science and pollution research international 2017-02, Vol.24 (6), p.5690-5700
• Main Authors: Da-Silva, E., Barthelmebs, L., Baudart, J.
• Format: Article
• Language: English
• Subjects: Aquatic Pollution; Assaying; Atmospheric Protection/Air Quality Control/Air Pollution; Bacteria; Bacteriology; Biotechnology; Chemiluminescence; Deoxyribonucleic acid; DNA; DNA probes; DNA, Bacterial - genetics; DNA, Viral; Earth and Environmental Science; Ecotoxicology; Environment; Environmental Chemistry; Environmental Engineering; Environmental Health; Environmental Monitoring - methods; Environmental science; Environmental Sciences; Hybridization; Laboratory equipment; Life Sciences; Limit of Detection; Linearity; Microbiology and Parasitology; Nucleic Acid Hybridization - methods; Polymerase chain reaction; Research Article; Ribonucleic acid; RNA; RNA, Ribosomal, 16S - genetics; rRNA 16S; Sensitivity and Specificity; Sequence Analysis, DNA; Stability analysis; Strains (organisms); Target detection; Vibrio; Vibrio - genetics; Waste Water Technology; Water Management; Water Pollution Control; Waterborne diseases; Working conditions
• ISSN: 0944-1344; 1614-7499
• DOI: 10.1007/s11356-016-8193-9

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 −1 of 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.