尽管存在识别混合物的准则,这些措施通常发生在分析的终点,并且是长期的。为了便于早期发现混合物,我们将高分辨率解链(HRM)混合物筛选试验整合到法医工作流程的qPCR步骤中,产生集成的QuantifilerTMTrio-HRM测定。化验,当与预测工具结合时,允许对样品的贡献者状态进行75.0%的准确识别(单一来源与混合物)。为了阐明开发的qPCR-HRM测定的局限性,进行了发育验证研究,评估重复性和不同DNA比率的样品,贡献者,和质量。从这项工作中,已确定集成QuantifilerTMTrio-HRM测定能够准确地鉴定具有多达五个供体的混合物和比例高达1:100的混合物。Further,最佳性能浓度范围为0.025至0.5ng/µL。有了这些结果,然后分析类似证据的DNA样本,导致100.0%的混合物样品被准确识别;此外,每次将样本预测为单一来源时,这是真的,给任何单一来源的电话提供信心。总的来说,QuantifilerTMTrio-HRM综合分析在qPCR阶段表现出增强的辨别混合样品与单一来源样品的能力,无论贡献者的性别如何。
Although guidelines exist for identifying mixtures, these measures often occur at the end-point of analysis and are protracted. To facilitate early mixture detection, we integrated a high-resolution melt (
HRM) mixture screening assay into the qPCR step of the forensic workflow, producing the integrated QuantifilerTM Trio-
HRM assay. The assay, when coupled with a prediction tool, allowed for 75.0% accurate identification of the contributor status of a sample (single source vs. mixture). To elucidate the limitations of the developed qPCR-
HRM assay, developmental validation studies were conducted assessing the reproducibility and samples with varying DNA ratios, contributors, and quality. From this work, it was determined that the integrated QuantifilerTM Trio-
HRM assay is capable of accurately identifying mixtures with up to five contributors and mixtures at ratios up to 1:100. Further, the optimal performance concentration range was found to be between 0.025 and 0.5 ng/µL. With these results, evidentiary-like DNA samples were then analyzed, resulting in 100.0% of the mixture samples being accurately identified; furthermore, every time a sample was predicted as a single source, it was true, giving confidence to any single-source calls. Overall, the integrated QuantifilerTM Trio-
HRM assay has exhibited an enhanced ability to discern mixture samples from single-source samples at the qPCR stage under commonly observed conditions regardless of the contributor\'s sex.