Simultaneous visual detection of single-nucleotide variations in tuna DNA using DNA/RNA chimeric probes and ribonuclease A

The need for detection of single-nucleotide polymorphisms (SNPs) is rapidly increasing for molecular diagnostics, species authentication, and food traceability. In many detection technologies, fluorescence probes have the advantage of simultaneous detection of multiple analytes using multiple color fluorescence dyes. In addition, an adequate concentration of fluorescence can be observed by the naked eye. We conducted a visual ribonuclease protection assay using multicolor fluorescence probes for the simultaneous detection of multiple tuna species. The assay includes amplification of a target RNA sequence by in vitro transcription, hybridization with DNA/RNA chimeric fluorescence probes, and cleavage of mismatched RNA bases by ribonuclease A. Fragmented dye-labeled oligonucleotides were easily removed by centrifugal gel filtration. Using three fluorescent probes, fluorescence signals related to the target SNP were simply found by the visual observation of eluates. Moreover, the dual fluorescence system was used for obtaining the mixing ratio of two tuna species. This technique appeared to be convenient for detection of interest species in food products.