Twenty-three years of PCR-based seafood authentication assay development: What have we learned?

Seafood is a prime target for fraudulent activities due to the complexity of its supply chain, high demand, and difficult discrimination among species once morphological characteristics are removed. Instances of seafood fraud are expected to increase due to growing demand. This manuscript reviews the application of DNA-based methods for commercial fish authentication and identification from 2000 to 2023. It explores (1) the most common types of commercial fish used in assay development, (2) the type of method used, (3) the gene region most often targeted, (4) provides a case study of currently published assays or primer-probe pairs used for DNA amplification, for specificity, and (5) makes recommendations for ensuring standardized assay-based reporting for future studies. A total of 313 original assays for the detection and authentication of commercial fish species from 191 primary articles published over the last 23 years were examined. The most explored DNA-based method was real-time polymerase chain reaction (qPCR), followed by DNA sequencing. The most targeted gene regions were cytb (cytochrome b) and COI (cytochrome c oxidase 1). Tuna was the most targeted commercial fish species. A case study of published tuna assays (n = 19) targeting the cytb region found that most assays were not species-specific through in silico testing. This was conducted by examining the primer mismatch for each assay using multiple sequence alignment. Therefore, there is need for more standardized DNA-based assay reporting in the literature to ensure specificity, reproducibility, and reliability of results. Factors, such as cost, sensitivity, quality of the DNA, and species, should be considered when designing assays.