Smith Chastyn, Williams Andrea L, Wines Hannah E, Cloudy Darianne C, Cox Jordan O, J Seashols-Williams Sarah, Boone Edward L, Green Tracey Dawson
Department of Forensic Science, Virginia Commonwealth University, 1015 Floyd Avenue, Richmond, VA, 23284, USA.
Integrative Life Sciences, Virginia Commonwealth University, 1000 Cary Street, Richmond, VA, 23284, USA.
Int J Legal Med. 2025 May;139(3):1045-1052. doi: 10.1007/s00414-025-03427-z. Epub 2025 Feb 1.
In recent years, DNA analysis techniques have drastically increased in sensitivity, allowing for low template DNA samples to be more easily detected and used for identification. Since the problems inherent in low template DNA are exacerbated in DNA mixture samples, it would be advantageous to incorporate an assay earlier in the DNA workflow that could detect a mixture and, potentially, determine the number of contributors. Some real-time PCR instruments have high-resolution melt curve analysis (HRM) capabilities, allowing for an opportunity to integrate an HRM screening assay into a commercial DNA quantification kit. This work describes the integration of a mixture screening HRM assay using STR loci D5S818 and D18S51 into Qiagen's Investigator Quantiplex kit. The integrated Quantiplex-HRM assay was tested on two qPCR platforms: The Rotor-Gene Q and the QuantStudio™ 6 Flex. Data from this assay was analyzed using linear discriminant and support vector machine analyses for sample classification. When HRM curve data from the Rotor-Gene Q was used for classification, the integrated assay exhibited an overall accuracy of 89.39%, correctly classifying 87.5% of single source samples and 100% of mixtures. When HRM curve data from the QuantStudio™ 6 Flex was used for classification, the integrated assay exhibited an overall accuracy of 87.88%, correctly classifying 87.5% of single source samples and 90% of mixtures. The overall accuracy of the integrated Quantiplex-HRM assay on both instruments met our goal of ≥ 80% accuracy, demonstrating the viability of the assay to detect mixtures when integrated into a commercial quantification kit.
近年来，DNA分析技术的灵敏度大幅提高，使得低模板DNA样本更容易被检测并用于鉴定。由于低模板DNA固有的问题在DNA混合样本中会加剧，因此在DNA工作流程中更早地纳入一种能够检测混合样本并可能确定贡献者数量的检测方法将是有利的。一些实时PCR仪器具有高分辨率熔解曲线分析（HRM）功能，这为将HRM筛选检测整合到商业DNA定量试剂盒中提供了机会。这项工作描述了将使用STR基因座D5S818和D18S51的混合样本筛选HRM检测整合到Qiagen公司的Investigator Quantiplex试剂盒中的过程。将整合后的Quantiplex-HRM检测在两个qPCR平台上进行了测试：Rotor-Gene Q和QuantStudio™ 6 Flex。使用线性判别分析和支持向量机分析对该检测的数据进行样本分类。当使用Rotor-Gene Q的HRM曲线数据进行分类时，整合后的检测总体准确率为89.39%，正确分类了87.5%的单一样本和100%的混合样本。当使用QuantStudio™ 6 Flex的HRM曲线数据进行分类时，整合后的检测总体准确率为87.88%，正确分类了87.5%的单一样本和90%的混合样本。整合后的Quantiplex-HRM检测在这两种仪器上的总体准确率达到了我们≥80%准确率的目标，证明了该检测整合到商业定量试剂盒中检测混合样本的可行性。
