Author: Amy Chan
Chan, Amy, 2024 Visualisation and Detection of Latent DNA in the Wildlife Trade: Pangolins as a Model Species, Flinders University, College of Science and Engineering
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This thesis detailed the detection and visualization of latent DNA deposited by pangolin scales using Diamond™ nucleic acid dye. Pangolins are the most illegally traded mammalian species with their scales commonly used in traditional medicines.
The ability of pangolin scales to shed cellular materials onto contact surfaces had not been investigated. In this project, the scales were removed from a roadkill Sunda pangolin (Manis javanica) and processed by drying to mimic the processing that pangolin scales undergo before distributing to the markets. A proof-of-concept study was first conducted using glass slides as a contact substrate. Two modes of cellular materials deposition, pressure, and friction were investigated and it was shown that more cellular materials were deposited onto the slides via friction than pressure. It was also deduced that much of the cellular materials deposited derived from the remnants of the dried tissues found on the ventral side of the scale as more cellular materials could be observed from the ventral side than the dorsal. DNA was then isolated from these cellular materials and identified to be of M. javanica origin using conventional PCR primers targeting the cyt b region of the mitochondrial DNA.
A M. javanica specific qPCR targeting the cyt b region of the mitochondrial DNA was also developed to quantify M. javanica latent DNA. The designed primer and probes set was determined to be specific by testing it against the DNA from M. javanica, S. gigantea, S. temminckii, P. tricuspis, P. tetradactyla and human. This qPCR was then used to compare the amount of latent M. javanica DNA recovered from glass slides using swabs or tape – lift and, extracted using commercial spin column or alkaline lysis DNA extraction method. It was found that swabbing was able to recover more DNA than tape – lifting. Swabbing followed by commercial spin column DNA extraction method also obtained the highest amount of DNA, indicating that this was the optimal workflow to be used when recovering and extracting latent M. javanica DNA from non – porous glass slides.
The experiment was then extended to recover latent M. javanica DNA from plastic bags used to store pangolin scales. M. javanica scales were packaged into five plastic bags and the presence of latent DNA was detected using Diamond™ Nucleic acid dye. Swabs were used to recover the stained cellular materials from various locations in the five bags, and isolated DNA was quantified using the established qPCR. As expected, a greater number of stained particles were found at the bottom of the bag than at the top. Latent M. javanica DNA were then recovered and extracted using a combination of swabbing or tape – lifting DNA recovery techniques followed by commercial spin column or alkaline lysis DNA extraction method and the qPCR results showed that using swabbing, followed by commercial spin column extraction method would recover and isolate the highest amount of latent M. javanica DNA deposited on the plastic bags, correlating well with the results obtained previously from glass slides.
Keywords: Wildlife forensic, species identification, latent DNA, pangolin
Subject: Biological Sciences thesis
Thesis type: Masters
Completed: 2024
School: College of Science and Engineering
Supervisor: Adrian Linacre