Real-time Loop-Mediated Isothermal Amplification (LAMP) Using Self-quenching Fluorogenic Probes: the Application in Skipjack Tuna (Katsuwonus pelamis) Authentication

Target-specific detection of LAMP amplicons can be achieved through the use of target-specific probes or modified primers as biorecognition elements, decreasing the likelihood of detecting false-positive signals. The present work selected skipjack tuna as a case study, and developed a novel LAMP assay coupled with the self-quenching fluorogenic probe for rapid detection of skipjack tuna. Specifically, the inner forward primer (FIP) was identified as the candidate primer to design the self-quenching fluorogenic probe. The fluorophore attached to the FIP (FIP-FAM) is self-quenched in unbound state. After binding to the dumbbell-shaped DNA target specifically, the FAM fluorophore is de-quenched, resulting in the fluorescence development. With the novel assay, as little as 5 fg of skipjack tuna, DNA could be detected. Moreover, the novel assay embraces a higher tolerance to impurities than non-specific fluorescent dyes, and succeeded the authentication of skipjack tuna from 6 commercial tuna products.