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

Abstract

1. 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.2. 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.3. 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 C using speciesspecific primer sets. 4. 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.5. 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.

A microfluidic fully paper-based analytical device integrated with loop-mediated isothermal amplification and nano-biosensors for rapid, sensitive, and specific quantitative detection of infectious diseases

“…The LAMP reaction produced a mixture of stem-loop DNA products of different sizes. 64,65 As shown in Fig. 4C, the specific ladder-pattern bands of LAMP products from N. meningitidis presented a mixture of DNA amplicons with various sizes due to loop-mediated amplification reactions, which confirmed the successful on-chip LAMP reactions using μFPAD to detect N. meningitidis .…”

Universal closed-tube barcoding for monitoring the shark and ray trade in megadiverse conservation hotspots

“…While existing genetic-based monitoring tools continue to be useful in many situations (Fields et al, 2015; Shivji et al, 2002)(Cardeñosa et al, 2018; Lin et al, 2021), FASTFISH-ID ™ seems poised to significantly expand the horizons of DNA-based control: alongside its speed, portability, and universality, the method exhibits single nucleotide resolution (Rice et al, 2014) which can minimize the risk of similar fluorescent signatures, particularly when more species are added to a reference library (Naaum et al, 2021). This is a particularly compelling argument for its implementation, as CITES lists are likely to continue to expand in the future.…”

Universal Closed-Tube Barcoding for Monitoring the Shark and Ray Trade in Megadiverse Conservation Hotspots

“…While existing genetic-based monitoring tools continue to be useful in many situations (Fields et al, 2015;Shivji et al, 2002) (Cardeñosa et al, 2018;Lin et al, 2021), FASTFISH-ID TM seems poised to significantly expand the horizons of DNAbased control: alongside its speed, portability, and universality, the method exhibits single nucleotide resolution (Rice et al, 2014) which can minimize the risk of similar fluorescent signatures, particularly when more species are added to a reference library (Naaum et al, 2021). This is a particularly compelling argument for its implementation, as CITES lists are likely to continue to expand in the future.…”

“…More recently, advances in real-time PCR have eliminated the sequencing stage, thereby allowing species identification to be conducted in the field. This approach uses target-specific primers and fluorescent dyes to detect the presence of the targeted nucleic acid template during PCR amplification and has been successfully applied to detect several CITES-listed shark species in a single run tube (Cardeñosa et al, 2018) and Multiplex LAMP (Lin et al, 2021). However, given their reliance on species-specific primers and probes, these methods are better suited to screening large numbers of specimens from one or few species rather than from a wide variety of species.…”