Establishment of a rapid method for skipjack tuna (Katsuwonus pelamis) authentication using molecular beacons in loop-mediated isothermal amplification

One major drawback to the traditional loop-mediated isothermal amplification (LAMP) detection methods is the increased likelihood of detecting false-positive signals derived from non-specific amplification. Molecular beacon (MB) is increasingly being used in many applications and the MB-LAMP assay has proved itself as a target-specific method. The present work selected skipjack tuna as a case study, and developed a novel MB-LAMP assay for rapid species authentication. Specifically, the optimal MB structure includes 13 nucleobases in the loop region (binding specifically to loop primer LF) and 5 nucleobases in the stem region. For the established MB-LAMP assay, in the presence of the amplicons, the MB probe LFP-1 hybridizes to its target and forms a double helix. The change in conformation separates the quencher from the fluorophore, thereby resulting in the fluorescence release. The novel MB-LAMP assay has proved its specificity and can detect as little as 0.5 pg of skipjack tuna DNA.