A quadruplex real-time qPCR assay for the simultaneous assessment of total human DNA, human male DNA, DNA degradation and the presence of PCR inhibitors in forensic samples: A diagnostic tool for STR typing

A quadruplex real-time qPCR assay was developed to simultaneously assess total human DNA, human male DNA, DNA degradation and PCR inhibitors in forensic samples. Specifically, the assay utilizes a ∼170–190bp target sequence that spans the TH01 STR locus to quantify total human DNA (nuTH01), a 137bp target sequence directly adjacent to the SRY gene to quantify human male DNA (nuSRY), a 67bp target sequence flanking the CSF1PO STR locus (nuCSF) to assess degradation (nuCSF:nuTH01 ratio) and a 77bp synthetic DNA template used as an internal PCR control target sequence (IPC) for the assessment of PCR inhibition. Validation studies, performed on an ABI 7500 SDS instrument using TaqMan® and TaqManMGB® detection, indicate each of the targets in the quadruplex assay performs effectively and is informative even when challenged with DNase-degraded and hematin-inhibited samples. The nuTH01–nuSRY–nuCSF–IPC quadruplex qPCR assay is envisioned to assist in the choice of the most informative DNA typing system available, which may include standard autosomal STR typing when the results indicate the presence of non-degraded, single gender DNA or non-degraded, male:female mixtures at ratios expected to yield probative alleles; Y STR typing in samples containing a male component that is overwhelmed by the presence of an excess of female DNA; reduced amplicon size STR typing (“MiniSTRs”) where the nuCSF:nuTH01 ratio indicates the sample is highly degraded; enhanced STR amplification with additional AmpliTaq Gold®/BSA and/or sample clean-up when the presence of PCR inhibitors is suggested by a delayed IPC CT value or mitochondrial DNA typing in samples where little to no nuclear DNA is detected. The present study includes evaluations of species specificity, sensitivity, precision, reproducibility, male–female mixtures, population samples and applications to various casework-type samples as indicated by the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines.