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Article Dans Une Revue Environmental Science and Pollution Research Année : 2017

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

Résumé

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
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hal-03943912 , version 1 (17-01-2023)

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Elise Da-Silva, L. Barthelmebs, J. Baudart. Development of a PCR-free DNA-based assay for the specific detection of Vibrio species in environmental samples by targeting the 16S rRNA. Environmental Science and Pollution Research, 2017, 24 (6), pp.5690-5700. ⟨10.1007/s11356-016-8193-9⟩. ⟨hal-03943912⟩
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