Rapid RNase H-dependent PCR lateral flow assay for the detection of red snapper

Red snapper (Lutjanus campechanus or Lutjanus purpureus) is one of the most commercially important and commonly mislabeled seafood species in the United States. Existing molecular methods for species identification are expensive and require being sent to an off-site laboratory, which takes multiple days to process. In this study, we have standardized a rapid rhPCR-coupled lateral flow assay for the identification of red snapper species. Our assay involved a simple heat-lysis DNA extraction procedure and a multiplex PCR reaction, consisting of a red snapper single nucleotide polymorphism (SNP)-specific rhPCR primer, thermotolerant RNase H2 enzyme, and an internal amplification control (IAC) for their onsite identification. Amplicons generated in the PCR reaction were detected using dual target lateral flow strips. The standardized assay was validated with 108 barcoded fish samples. Samples identified as Lutjanus campechanus or L. purpureus by DNA barcoding formed three distinct bands, while other fish species formed only two bands on the lateral flow strips. A minimum of 0.37 ng of crude DNA was detectable in the rhPCR reaction and generated a visible band on the lateral flow dipstick. The assay showed 100% specificity and took 90-120 min to complete. The assay generated results that can be used to authenticate red snapper at the wholesale and retail levels of seafood processing and commerce. Our results confirm the ability of rhPCR-lateral flow assays to be an alternative tool for onsite species authentication for the seafood industry.