Development of a triplex mtDNA qPCR assay to assess quantification, degradation, inhibition, and amplification target copy numbers.

A hybrid absolute/relative qPCR assay which provides information regarding the condition of mitochondrial DNA (mtDNA) in a DNA sample is described. MtDNA concentration (copy number/μL) is determined via absolute quantification using a standard curve of a synthetic duplex DNA previously described (Kavlick et al., 2011). The state of mtDNA degradation is determined via the relative quantification of a mtDNA target found within the 16 s rRNA gene which is 3× longer than that of the short target in the former duplex assay, using the delta, delta Ct (ΔΔCt) method. The presence or absence of PCR inhibitors in the sample is qualitatively determined using a custom internal positive control (IPC) system which targets a unique and non-naturally occurring duplex DNA sequence. This IPC effectively detected inhibition by humic acid, tannic acid, melanin, and EDTA. All three assay components utilize sensitive and specific hydrolysis probes. The utility of ΔΔCt method was demonstrated in a series of experiments involving laboratory-fragmented DNA. Also described is a method for estimating copy number of any mtDNA target longer than the two targets amplified. The described triplex assay works well for intact and for fragmented or degraded mtDNA and therefore may be useful in forensic and ancient DNA disciplines as well as in biomedical research or practice. Published by Elsevier B.V.