Development and validation of ultra-fast quantitative real-time PCR method to differentiate between Oncorhynchus keta and Oncorhynchus mykiss

Abstract

The ultra-fast quantitative real-time polymerase chain reaction (qPCR) assay was developed and validated to differentiate the morphologically similar ones, Oncorhynchus keta and Oncorhynchus mykiss. Species-specific primers were designed for the COI genes of mtDNA. The species-specific primers designed for O. keta and O. mykiss were selectively amplified by O. keta and O. mykiss DNA, respectively. The sensitivity of O. keta and O. mykiss primers was 1 ng/μL. Quantitative testing showed that the results met the ‘Guidelines on Standard Procedures for Preparing Analysis Method such as Food’ proposed by the Ministry of Food and Drug Safety. The qPCR method developed and validated in this study for identifying O. keta and O. mykiss has advantages such as speed and field applicability. Therefore, this method is expected to help control forgery and alteration of raw materials in the seafood industry.