An Assessment of the Performance Limitations of the Integrated Quantifiler™ Trio-HRM Assay: A Forensic Tool Designed to Identify Mixtures at the Quantification Stage

• NCJ Number: 309476
• Journal: Genes Volume: 15 Issue: 6 Dated: June 2024 Pages: 768
• Author(s): Chastyn Smith; Sarah J. Seashols-Williams; Edward L. Boone; Tracey Dawson Green
• Date Published: June 2024
• Length: 14 pages
• Grant Number(s): 2019-DU-BX-0003
• Agencies: NIJ-Sponsored
• Publication Type: Research (Applied/Empirical)
• Award: Towards Commercialization: Preliminary developmental validation of a high resolution melt curve mixture prediction assay and SVM tool

Annotation

This article describes the materials, methodology, and results of a research study that demonstrates how the integrated the Quantifiler™ Trio-HRM assay is an accurate, inexpensive, and reliable way to gain pertinent information about a sample’s contributor status, early in the analysis workflow; the authors suggest that the tool can provide forensic examiners with an effective way to screen and triage evidence items before the end-point of analysis.

Abstract

Although guidelines exist for identifying mixtures, these measures often occur at the end-point of analysis and are protracted. To facilitate early mixture detection, the authors integrated a high-resolution melt (HRM) mixture screening assay into the qPCR step of the forensic workflow, producing the integrated Quantifiler™ Trio-HRM assay. The assay, when coupled with a prediction tool, allowed for 75.0 percent 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 Quantifiler™ 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 Quantifiler™ 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. (Published Abstract Provided)