Integration of a high-resolution melt curve assay into a commercial quantification kit for preliminary identification of biological mixtures.

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.