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 2.5nM of PC, and detection limit was determined as 0.1nM. In a second step, specificity was checked by using RNA extracted from a panel of 31 environmental bacterial strains. Detection limit of 5ngμL-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 30days. The use of the pre-made plates enables the assay to be completed in 2h. The developed assay appeared as an interesting tool to determine the presence of bacteria in environmental samples.