A direct multiplex loop-mediated isothermal amplification method to detect three CITES-listed shark species

Abstract

Species identification of sharks under catch or trade regulations is important for law enforcement and species conservation. Rapid detection of Convention on the International Trade in Endangered Species (CITES)-listed species is needed for on-site screening. Species-specific primers were designed to target three mitochondrial genes (ND2, COI, and CytB) in both the simplex and multiplex loop-mediated isothermal amplification (LAMP) assay for the pelagic thresher shark (Alopias pelagicus), the bigeye thresher shark (Alopias superciliosus), and the scalloped hammerhead shark (Sphyrna lewini), respectively. Another primer set designed to target S. lewini was used for detection-limit testing of the LAMP assay. The refined direct multiplex LAMP was used to detect the three CITES-listed shark species and omitted the lengthy DNA extraction process. A homogenizer was used to release the DNA from the shark tissues, and a simplex or multiplex LAMP reaction was conducted for 30 min in an incubator at 65 degrees C using species-specific primer sets. Positive LAMP reactions showed a colour change from pink to yellow, whereas negative reactions showed no colour change. Multiplex LAMP assays were performed using 84 samples, which successfully identified the target and non-target species and provided a fast (<1 h), simple, and reliable method to distinguish three CITES-listed shark species from the other non-target species, for either fresh or dry fin products. Results of this study and the method developed will play a critical role in assisting fishery agencies and customs officials in identifying the illegal catch and trade of CITES-listed shark species.