9 research outputs found
Genetic identification of three CITES-listed sharks using a paper-based Lab-on-a-Chip (LOC)
Threatened shark species are caught in large numbers by artisanal and commercial fisheries and traded globally. Monitoring both which shark species are caught and sold in fisheries, and the export of CITES-restricted products, are essential in reducing illegal fishing. Current methods for species identification rely on visual examination by experts or DNA barcoding techniques requiring specialist laboratory facilities and trained personnel. The need for specialist equipment and/or input from experts means many markets are currently not monitored. We have developed a paper-based Lab-on-a-Chip (LOC) to facilitate identification of three threatened and CITES-listed sharks, bigeye thresher (Alopias superciliosus), pelagic thresher (A. pelagicus) and shortfin mako shark (Isurus oxyrinchus) at market source. DNA was successfully extracted from shark meat and fin samples and combined with DNA amplification and visualisation using Loop Mediated Isothermal Amplification (LAMP) on the LOC. This resulted in the successful identification of the target species of sharks in under an hour, with a working positive and negative control. The LOC provided a simple “yes” or “no” result via a colour change from pink to yellow when one of the target species was present. The LOC serves as proof-of-concept (PoC) for field-based species identification as it does not require specialist facilities. It can be used by non-scientifically trained personnel, especially in areas where there are suspected high frequencies of mislabelling or for the identification of dried shark fins in seizures
DNA capture efficiency of Whatman© glass microfiber filters (GF/C).
The DNA solution was made up to 25 ÎĽL with 5M GuHCl in concentrations ranging from 8 to 64 ng/ÎĽL. The maximum retention of the GF/C filters were recorded.</p
Elasmobranch and teleost fish species collected in Ecuador, USA and in captivity (Aquarium France as well as the percentage (%) match from Sanger Sequencing for both Forward (F) and Reverse (R) sequences for each species.
Elasmobranch and teleost fish species collected in Ecuador, USA and in captivity (Aquarium France as well as the percentage (%) match from Sanger Sequencing for both Forward (F) and Reverse (R) sequences for each species.</p
Fig 1 -
a) Overview of the procedure for cell lysis from a small piece of meat or wet fin sample taken from markets; b) Photograph showing the paper LOC design with five different coloured areas (panels) that was folded in a origami-style manner to enable different steps of the genetic analysis to be performed; c) Schematic showing the folding of the LOC for DNA binding and washing steps; d) Schematic showing the alternative folding of the LOC for DNA elution; e) Location of the five LAMP chambers for species-specific amplification, as well as positive and negative controls.</p
S1 Appendix -
Threatened shark species are caught in large numbers by artisanal and commercial fisheries and traded globally. Monitoring both which shark species are caught and sold in fisheries, and the export of CITES-restricted products, are essential in reducing illegal fishing. Current methods for species identification rely on visual examination by experts or DNA barcoding techniques requiring specialist laboratory facilities and trained personnel. The need for specialist equipment and/or input from experts means many markets are currently not monitored. We have developed a paper-based Lab-on-a-Chip (LOC) to facilitate identification of three threatened and CITES-listed sharks, bigeye thresher (Alopias superciliosus), pelagic thresher (A. pelagicus) and shortfin mako shark (Isurus oxyrinchus) at market source. DNA was successfully extracted from shark meat and fin samples and combined with DNA amplification and visualisation using Loop Mediated Isothermal Amplification (LAMP) on the LOC. This resulted in the successful identification of the target species of sharks in under an hour, with a working positive and negative control. The LOC provided a simple “yes” or “no” result via a colour change from pink to yellow when one of the target species was present. The LOC serves as proof-of-concept (PoC) for field-based species identification as it does not require specialist facilities. It can be used by non-scientifically trained personnel, especially in areas where there are suspected high frequencies of mislabelling or for the identification of dried shark fins in seizures.</div
LAMP primers for bigeye thresher shark (<i>Alopias superciliosus</i>), pelagic thresher shark (<i>A</i>. <i>pelagicus</i>) and shortfin mako shark (<i>Isurus oxyrinchus</i>).
LAMP primers for bigeye thresher shark (Alopias superciliosus), pelagic thresher shark (A. pelagicus) and shortfin mako shark (Isurus oxyrinchus).</p
DNA from fin and muscle tissue samples was purified using 70% ethanol in 5 ÎĽL increments.
The protein concentrations of the resulting samples were compared. The red dashed line indicates the plateau in protein concentration at 0.1 ng/ÎĽL when 60 ÎĽL of ethanol is loaded onto the LOC.</p
Fig 4 -
Schematic (top panel) and photographic (bottom panel) examples of LOC results showing amplification of target species (a) bigeye thresher shark (Alopias superciliosus), (b) pelagic thresher shark (A. pelagicus), and (c) shortfin mako shark (Isurus oxyrinchus), and no amplification of non-target species (d) blue shark (Prionace glauca).</p