Discrimination of Thunnus thynnus orientalis and T. thynnus thynnus using double-fluorescence FRIP assay

A combination of double fluorescence-labeled probes was applied to multiplex discrimination of Thunnus thynnus orientalis (Pacific bluefin) and T. thynnus thynnus (Atlantic bluefin) in the fluorogenic ribonuclease protection (FRIP) assay. The FRIP assay provides information about the absence or presence of known single nucleotide polymorphisms (SNPs). It requires only one pair of fluorescence donor- and acceptor probes for discriminating one species, and allows rapid and sensitive analysis. DNA was extracted from individuals of six Thunnus species and subjected to PCR with primers targeting the T7 RNA polymerase promoter region, and then in vitro transcribed RNA was produced. Both an FITC-labeled probe targeting Pacific bluefin tuna and a TAMRA-labeled probe targeting Atlantic bluefin tuna were simultaneously hybridized to individual RNA samples in hybridization buffer solution. Fluorescence emissions were temporary quenched via FRET, but fluorescence intensity recovered in the response to the presence of mismatches in DNA- RNA hybridridization following RNase A digestion. Similar results were also observed in visualization of FRIP reaction mixtures under UV light excitation. The FRIP assay, which is a simple, rapid, sensitive and economic analytical method, can be employed in a variety of food inspection and authentication applications.