Kitpipit Thitika, Penchart Kitichaya, Ouithavon Kanita, Satasook Chutamas, Linacre Adrian, Thanakiatkrai Phuvadol
Forensic Science Program, Department of Applied Science, Faculty of Science, Prince of Songkla University, Thailand.
Wildlife Forensic Science Unit, Department of National Parks, Wildlife and Plant Conservation, Thailand.
Forensic Sci Int. 2016 Oct;267:210-217. doi: 10.1016/j.forsciint.2016.08.037. Epub 2016 Sep 12.
Demand for ivory and expansion of human settlements have resulted in a rapid decline in the number of elephants. Enforcement of local and international laws and regulations requires identification of the species from which any ivory, or ivory products, originated. Further geographical assignment of the dead elephant from which the ivory was taken can assist in forensic investigations. In this study, a real-time PCR assay using melt curve analysis was developed and fully validated for forensic use. The presence or absence of three Elephantidae-specific and elephant species-specific melting peaks was used to identify the elephant species. Using 141 blood and ivory samples from the three extant elephant species, the assay demonstrated very high reproducibility and accuracy. The limit of detection was as low as 0.031ng of input DNA for conventional amplification and 0.002ng for nested amplification. Both DNA concentrations are typically encountered in forensic casework, especially for degraded samples. No cross-reactivity was observed for non-target species. Evaluation of direct amplification and nested amplification demonstrated the assay's flexibility and capability of analyzing low-template DNA samples and aged samples. Additionally, blind trial testing showed the assay's suitability application in real casework. In conclusion, wildlife forensic laboratories could use this novel, quick, and low-cost assay to help combat the continuing poaching crises leading to the collapse of elephant numbers in the wild.
对象牙的需求以及人类定居点的扩张导致大象数量迅速减少。执行地方和国际法律法规需要确定任何象牙或象牙制品的来源物种。对获取象牙的死亡大象进行进一步的地理定位有助于法医调查。在本研究中，开发了一种使用熔解曲线分析的实时PCR检测方法，并进行了全面验证以供法医使用。通过三种象科特异性和大象物种特异性熔解峰的有无来鉴定大象物种。使用来自三种现存大象物种的141份血液和象牙样本，该检测方法显示出非常高的重现性和准确性。常规扩增的检测限低至0.031 ng输入DNA，巢式扩增的检测限低至0.002 ng。这两种DNA浓度在法医案件工作中都很常见，尤其是对于降解样本。未观察到非目标物种的交叉反应。直接扩增和巢式扩增的评估证明了该检测方法在分析低模板DNA样本和陈旧样本方面的灵活性和能力。此外，盲法试验测试表明该检测方法适用于实际案件工作。总之，野生动物法医实验室可以使用这种新颖、快速且低成本的检测方法来帮助应对持续的偷猎危机，这种危机正导致野生大象数量的崩溃。
