Refinement and application of 12S rRNA meta-barcoding primers for seafood identification in multispecies product

Fish balls, processed from fish surimi, are a popular seafood product that has become a focus of food adulteration monitoring. To reveal the ingredients in fish balls and ensure authenticity, a set of meta-barcoding primers targeting the 12S rRNA gene was designed and optimized using mixed DNA samples from fish, livestock, and poultry, which are common ingredients in fish ball production. PCR products were sequenced using Next-Generation Sequencing (NGS) technology to identify animal-derived species. The results demonstrated that the primers accurately identified all mixed ingredients in various proportions. Nine batches of commercial fish balls were analyzed for species composition using the designed primers. All samples contained multiple fish species, with two batches showing serious discrepancies between actual ingredients and labeling. In conclusion, the optimized 12S rRNA meta-barcoding primers effectively identified the ingredients in multi-species fish balls, offering a complementary DNA barcoding tool for market regulation and consumer protection.