Comparative evaluation of conventional, real-time, and ultrafast real-time PCR assays for accurate identification of...

In this study, a species-specific primer was developed for the cytochrome c oxidase subunit I (COI) region to identify krill species in processed foods. Optimal conditions for qualitative conventional PCR, real-time PCR (RT-PCR), and ultrafast RT-PCR were established by adjusting annealing temperature, primer concentration, and annealing time. Specificity and sensitivity were established by adjusting annealing temperature, primer concentration, and annealing time. Specificity and sensitivity were established by analyzing the presence or absence of a specific band and the limit of detection. The concentration was validated as 0.001 ng/μL for RT-PCR and ultrafast RT-PCR as well as 0.005 ng/μL for conventional PCR. All validation items, including false-negative and false-positive rates, were performed according to the Codex Alimentarius guidelines. Among 14 commercial products tested, E. pacifica was identified in a salted fermentation product of Korean origin through ultrafast RT-PCR, offering a rapid, sensitive tool for regulatory seafood authentication.