Abstract
The present study aimed to develop a novel loop-mediated isothermal amplification (LAMP) assay targeting cytochrome b gene for rapid screening of skipjack tuna, the species with the largest output among the Thunnini tribe. In particular, LAMP primers specific for skipjack tuna were designed and the specificity was confirmed against 22 other fish species. The LAMP assay was optimized, and the highest efficiency of the real time fluorescence LAMP determination was obtained by adding inner primers of 1.6 μM, outer primers of 0.2 μM, and 5 × Evagreen. The LAMP assay could detect as low as 50 pg skipjack tuna DNA, by both colorimetric and real time fluorescent determination. SDS-Proteinase K method was selected as the most feasible method for the extraction of high quantity and quality DNA from tuna products. Skipjack tuna can be detected from only four canned tuna products using the developed LAMP assay, cross-confirmed by DNA sequencing method. Finally, at least eight products can be considered as mislabeling, due to containing no Scombridae species. Therefore, with specificity and sensitivity, the novel LAMP method can be used for rapid screening of skipjack tuna in processed fish products.
Published Version
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