A rapid rhPCR-lateral flow assay for authenticating royal red and Argentine red shrimp in the U.S. seafood supply chain.

In the United States, approximately one-third of shrimp sold are misrepresented. DNA barcoding requires robust infrastructure and one to three days to generate results, limiting enforcement utility. We hypothesized that RNase H2-dependent PCR (rhPCR) primers targeting cytochrome oxidase subunit I (COI) sequence-specific sites, coupled with lateral flow detection, could enable rapid species authentication of royal red shrimp (Pleoticus robustus) and Argentine red shrimp (Pleoticus muelleri). Species-specific rhPCR primers labeled with FAM and biotin were designed and evaluated using crude DNA extracts from 65 barcoded shrimp specimens representing seven commercially traded species. The assays were further validated using 20 blinded retail samples marketed as royal red shrimp. Both assays demonstrated high specificity comparable to DNA barcoding results, and an analytical sensitivity of 0.01 ng/μL. Analysis of retail samples revealed 60% of samples were not royal red shrimp. Total time-to-results for 20 samples was approximately 150 min, demonstrating a rapid, simpler, and sequencing-free alternative for regulatory enforcement.