Rapid identification of Atlantic cod, sablefish, and toothfish using recombinase polymerase amplification combined with lateral flow dipstick: Application in the detection of cod-related fish products in China

Gadus species are highly valued for their superior nutritional quality ; however, frequent mislabeling raises consumer concerns. Sablefish ( Anoplopoma fimbria ) and toothfish ( Dissostichus eleginoides and D. mawsoni ) are often marketed in China under names that include “cod” for commercial reasons. This study developed a recombinase polymerase amplification assay (RPA) combined with a lateral flow dipstick (LFD) to rapidly detect Atlantic cod ( Gadus morhua ), sablefish, and toothfish. Two specific primers (including one biotin-labeled reverse primer) and a probe with 5′ FAM label and THF-substituted thymine were designed for each species, targeting the interspecific regions of the cytochrome c oxidase subunit I ( COI ) gene. The developed RPA-LFD assays were specific for Atlantic cod, sablefish, and toothfish, showing no cross-reactivity with other teleost species. The assays demonstrated detection limits of 1–100 copies/μL for recombinant plasmid DNA and 1–100 fg/μL for genomic DNA across Atlantic cod, sablefish, and Patagonian toothfish. Primer concentrations were uniformly optimized to 0.4 μM, while probe concentrations were set at 0.12 μM for Atlantic cod and Patagonian toothfish, and 0.18 μM for sablefish. RPA conditions varied slightly, with optimal durations of 14–16 min and temperatures of 36–39 °C tailored to each species. Testing of 67 frozen cod-related fish products from large retail stores in China showed 100 % concordance with COI sequencing. Additionally, the results revealed a high mislabeling rate of 17.91 % among these products, although the substituted species were relatively limited, primarily including Pacific cod ( M . poutassou ), Alaska pollock ( G . chalcogrammus ), and blue whiting ( Micromesistius poutassou ). This RPA-LFD assay is rapid, simple, visual, sensitive, and accurate for identifying Atlantic cod, sablefish, and toothfish, and could help monitor cod-related fish adulteration on site. • RPA-LFD assay developed for identifying Atlantic cod, sablefish, and toothfish. • Assay demonstrated detection limits of 1–100 fg/μL gDNA with high specificity. • RPA-LFD showed 100 % concordance with COI sequencing in 67 codfish sample tests. • 23.88 % mislabeling in cod from China's stores, replaced by pollock or blue whiting. • RPA-LFD offers a fast, sensitive, and accurate tool for monitoring cod adulteration.