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

## TL;DR

A new rapid test was developed to identify New Zealand green-lipped mussels and distinguish them from similar species, even after cooking.

## Contribution

A novel dRAA-LFD assay was developed for on-site authentication of Perna canaliculus against Perna viridis.

## Key findings

- The assay achieved optimal performance at 40°C for 30 minutes with no cross-reactivity.
- Cooked mussel samples remained identifiable using the dRAA-LFD assay.
- 13 out of 15 commercial products were identified as P. canaliculus using the test.

## Abstract

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.