Developmental Validation of the Quantifiler^® Duo DNA Quantification Kit for Simultaneous Quantification of Total Human and Human Male DNA and Detection of PCR Inhibitors in Biological Samples*

Abstract

The Quantifiler Duo DNA Quantification kit enables simultaneous quantification of human DNA and human male DNA as well as detection of inhibitors of PCR in a single real-time PCR well. Pooled human male genomic DNA is used to generate standard curves for both human (ribonuclease P RNA component H1) and human male (sex determining region Y) specific targets. A shift in the cycle threshold (C(T)) values for the internal positive control monitors the presence of PCR inhibitors in a sample. The assay is human specific and exhibits a high dynamic range from 0.023 to 50 ng/microL. In addition, the multiplex assay can detect as little as 25 pg/microL of human male DNA in the presence of a 1000-fold excess of human female DNA. The multiplex assay provides assessment of the DNA extract and guidance for the selection of the appropriate AmpFlSTR Amplification Kit to obtain interpretable short tandem repeat profiles.

Safeguarding forensic DNA reference samples with nullomer barcodes

“…34,35 Internal positive controls are already an important part of forensic genotyping and qPCR quantification kits. 20,[23][24][25][26][27][28][29][30] In those kits, the IPC allows the forensic analyst to assess the presence of contaminating PCR inhibitors, such as hematin or humic acids. 25 Unlike our nullomer tagging protocol, current tags are present in the PCR reaction components, and so cannot distinguish reference from evidentiary samples.…”

“…Our results show that nullomer sequences can be used as an IAC, and as molecular tags and barcodes, successfully integrated into the multiplex PCR reactions of commercially available forensic profiling kits, and used along with PCR for sequencing. The use of IACs (sometimes designated internal positive controls, IPCs) is already commonplace for food and clinical microbiological testing, 19 forensic quantification kits, 20 , forensic human identification kits 21,22,23 , and are particularly useful for detecting PCR inhibitors. 24 For PCR-based tests of food-borne pathogens, the European Standardization Committee has developed guidelines that require the presence of an IAC.…”

Quantifiler® Duo DNA Quantification Kit: A Guiding Tool for Short Tandem Repeat Genotyping of Forensic Samples

“…The standard deviation values for the RPPH1, SRY, and IPC targets across all 6 plates ranged from 0.23 to 0.58, 0.15 to 0.63, and 0.17 to 0.35, respectively (data not shown). The data showed that at lower DNA concentrations, the standard deviations increased, most likely due to stochastic effects [12].…”

“…Increased C T values for the IPC target indicate the extent of the presence of PCR inhibitor(s). The results from the samples containing humic acid are described earlier [12].…”

Population genetic data for 17 Y STR markers from Benghazi (East Libya)

“…Since the beginning of the nineties (1992), when Lutz Roewer and colleagues described the first polymorphic Y-chromosome marker Y-27H39 -now better known as the STR locus DYS19 [3] the field of forensic Y chromosome analysis has been successfully developed [4]. National DNA databases collectively house millions of STR profiles around the world [5]. Global population databases have been established [6] yet studies focusing on Middle Eastern and North African countries are still rare.…”