We report on the detection and visualisation of latent DNA from pangolin scales deposited onto a plastic packaging material through the use of a nucleic acid staining dye. This latent DNA deposited by pangolin scales was subsequently isolated and analysed using DNA barcoding method. Pangolins are the most illegally traded mammalian species due to the demand for their scales and meat. The demand for their scales were mostly fuelled by its use in traditional medicines. The scales are usually packed into bags and transported globally via sea routes. This is the first report detailing the detection of trace latent DNA from processed wildlife products, on surfaces of bags that they were packaged in. Prior to this report, it was not known if the dried pangolin scales contained transferable quantities of biological material for DNA analyses. To address this, scales were removed from a roadkill Sunda pangolin (Manis javanica), processed by drying and packaged into one of five plastic bags. The presence of pangolin latent DNA was detected on the surface of the plastic bags and visualised using Diamond™ nucleic acid dye. Swabs were then used to recover the stained biological material from various locations in the five bags. The DNA was isolated and quantified using a newly designed quantitative PCR (qPCR) specific to M. javanica to amplify a fragment of the mitochondrial DNA cytochrome b gene. There was a positive correlation between the number of stained particles and DNA quantity, and a greater number of stained particles were found at the bottom of the bag than were found at the top. Conventional PCR targeting part of the cyt b gene amplified a product from all 30 samples taken from the bags and in all cases, sequence data generated matched that of the Sunda pangolin, as expected. All negative controls yielded no results. The method described here is the very first use of a nucleic acid staining dye to detect latent DNA from a mammalian species, other than humans, and highlights the opportunity for further use of Diamond™ nucleic acid dye in wildlife forensic science. It is anticipated that this method will be invaluable in retrieving latent DNA deposited by wildlife products from the environment in which they were contained, to determine the presence of these illegal wildlife products even when previously hidden, inaccessible, or no longer present physically. Further research is required to understand if the use on non-human mammalian wildlife species is feasible.

我们报告了通过使用核酸染色染料对沉积在塑料包装材料上的穿山甲鳞片中的潜在 DNA 进行检测和可视化。随后使用DNA 条形码方法分离并分析了穿山甲鳞片沉积的潜在 DNA 。由于对穿山甲鳞片和肉的需求，穿山甲是非法交易最多的哺乳动物物种。对它们的鳞片的需求主要是由于其在传统药物中的使用而推动的。秤通常装入袋子中并通过海上航线运输到全球。这是第一份详细介绍从加工野生动物产品的包装袋表面检测出微量潜在 DNA 的报告。在此报告发布之前，尚不清楚干穿山甲鳞片中是否含有可转移数量的用于 DNA 分析的生物材料。为了解决这个问题，我们从被路杀的马来穿山甲 ( Manis javanica )身上去除鳞片，进行干燥处理，然后包装到五个塑料袋之一中。在塑料袋表面检测到穿山甲潜在 DNA 的存在，并使用 Diamond™ 核酸染料进行可视化。然后用拭子从五个袋子的不同位置回收染色的生物材料。使用新设计的爪哇分枝杆菌特异性定量 PCR (qPCR) 来分离和定量 DNA，以扩增线粒体 DNA 细胞色素b基因的片段。染色颗粒数量与DNA数量呈正相关，袋子底部的染色颗粒数量多于顶部。传统的 PCR 靶向cyt b基因的一部分，扩增了从袋子中取出的所有 30 个样本的产物，并且在所有情况下，生成的序列数据都与预期的巽他穿山甲的序列数据相匹配。所有阴性对照均未产生结果。这里描述的方法是首次使用核酸染色染料来检测人类以外的哺乳动物物种的潜在 DNA，并强调了 Diamond™ 核酸染料在野生动物法医学中进一步使用的机会。预计该方法对于从野生动物产品所在环境中检索野生动物产品沉积的潜在 DNA 至关重要，以确定这些非法野生动物产品的存在，即使这些非法野生动物产品以前隐藏、无法接近或不再存在。需要进一步的研究来了解在非人类哺乳动物野生动物物种上的使用是否可行。