Duplex Recombinase-Aided Amplification-Lateral Flow Dipstick (dRAA-LFD) Assay for New Zealand Green-Lipped Mussel...

New Zealand green-lipped mussel (Perna canaliculus) is a premium seafood product that may be substituted with morphologically similar mussels after shucking and cooking, particularly Asian green mussel (Perna viridis). This study developed a rapid, on-site duplex recombinase-aided amplification-lateral flow dipstick (dRAA-LFD) assay to authenticate P. canaliculus and differentiate it from P. viridis. Species-specific primers were designed from mitochondrial COI alignment and combined in a dRAA reaction. Reaction conditions were optimized at 37-42 °C and 15-60 min. Specificity was assessed against 11 non-target seafood species, and sensitivity was evaluated using 2-fold serial dilutions. The assay was further validated using DNA from boiled (85 °C, 5-15 min), steamed (105 °C, 10-30 min), and fried (185 °C, 30-90 s) mussels, and 15 restaurant products labeled as New Zealand mussel dishes. Optimal performance was achieved at 40 °C for 30 min, with no cross-reactivity. The LFD detection limits were 0.05 ng/reaction for P. viridis and 0.2-0.1 ng/reaction for P. canaliculus. All cooked samples remained identifiable, and commercial testing classified 13/15 products as P. canaliculus and 2/15 as P. viridis. Overall, the dRAA-LFD assay enables rapid, equipment-light authentication of cooked mussel products for routine screening.