Demand for bear bile, a prized component of traditional Asian medicines, threaten Asiatic and sun bear population sustainability. While laws exist to prevent poaching and trafficking of bear parts and derivatives, smuggling persists with demand extending to surrogate species, including American black bears (Ursus americanus). Mitochondrial DNA (mtDNA) sequencing can identify products putatively containing biological bear material but can be undermined by PCR inhibitors in bile and a lack of sensitivity at trace levels. Quantitative PCR (qPCR) assays can be used to distinguish between closely related target species, while concomitantly evaluating inhibition and false negative results in low quality/quantity DNA applications. Herein, we develop a multiplexed qPCR assay to detect and differentiate among bear species, including highly diluted bile samples mixed within liquors as common dilutants. The assay detects as little as 10 locus copies/reaction of bear DNA with 95% confidence, distinguishing among sun, Asiatic and American black bears. Demonstrating the sensitivity and applicability of this assay in context of current bile mixture recipes, dilutions of 1:5,000 bile with ethanol, red wine, and spirits, all yielded clear quantifiable detections, where our data suggests as little as 1 drop of bile per 750 mL bottle of alcohol would still exceed the limits of detection (e.g., 1:15000 dilution or <0.05 mL bile per 750 mL bottle). Overall, this study provides a rapid, sensitive, and specific test to identify and distinguish among bear species commonly used for bile production to aid wildlife enforcement applications.

对熊胆（亚洲传统药物的珍贵成分）的需求威胁着亚洲熊和马来熊种群的可持续性。尽管有法律禁止偷猎和贩运熊的身体部位和衍生物，但走私现象依然存在，需求已扩展到替代物种，包括美洲黑熊 ( Ursus americanus )。线粒体 DNA (mtDNA) 测序可以识别假定含有生物熊物质的产品，但可能会受到胆汁中 PCR 抑制剂的破坏，并且在痕量水平上缺乏敏感性。定量 PCR (qPCR) 检测可用于区分密切相关的目标物种，同时评估低质量/数量 DNA 应用中的抑制和假阴性结果。在此，我们开发了一种多重 qPCR 检测方法来检测和区分熊的种类，包括作为常见稀释剂混合在液体中的高度稀释的胆汁样本。该检测以 95% 的置信度检测到熊 DNA 的每个基因座拷贝数少至 10 个，区分太阳黑熊、亚洲黑熊和美洲黑熊。在当前胆汁混合物配方、用乙醇、红酒和烈酒稀释 1:5,000 胆汁的背景下，证明了该测定的灵敏度和适用性，所有这些都产生了清晰的可量化检测，其中我们的数据表明每 750 份胆汁中的胆汁含量低至 1 滴mL 瓶酒精仍会超出检测限（例如，1:15000 稀释或每 750 mL 瓶 <0.05 mL 胆汁）。总体而言，这项研究提供了一种快速、灵敏且具体的测试，以识别和区分通常用于产生胆汁的熊种，以帮助野生动物执法应用。