Testing Candidate DNA Quantitation Standards with Several Real-Time Quantitative PCR Methods

Numerous real-time quantitative PCR (qPCR) methods have been developed in the last several years for use with forensic DNA samples. Ten different qPCR methods were used to evaluate DNA samples distributed in the NIST Interlaboratory DNA Quantitation Study 2004 (QS04) [1]. The target DNA concentrations of the QS04 samples were from 1.5 ng/μL to 50 pg/μL. About one-fifth of all QS04 results came from qPCR methods. These data show differences among the qPCR methods, both with regard to precision and bias. It is unclear from these data whether the observed differences are inherent to the methods or reflect differences in the standards used in their calibration. We here present our evaluation of several qPCR methods using six different human DNA calibration materials. All of the qPCR methods evaluated are either commercially available or have been published recently. Three of the calibration materials used in the evaluation are commercially available; three are derived from in-house purified single-donor blood samples. This study was designed to help direct development of candidate Standard Reference Material 2372, Human Genomic DNA Quantitation Standard.