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P066 – Absolute Quantification of Mitochondrial DNA Copy Number Using Multiplex Droplet Digital PCR for Forensic Applications
Forensic DNA analysis frequently begins with suboptimal sample quality and quantity. Degraded samples, inhibition, and low template input can all undermine confidence in quantification and downstream interpretation. Digital PCR offers a unique approach of DNA quantification. By segmenting samples into thousands of discrete reactions, dPCR facilitates absolute quantification without the need for external standards, enhances repeatability across laboratories, and augments tolerance to inhibitors frequently present in forensic samples.
The aim of this study was to transfer the Kavlicks primer set [1] from qPCR to ddPCR to quantify mtDNA and compare their performance using both methods.
The primers developed by Kavlick for a triplex mtDNA qPCR assay were adapted for use on the QIAcuity (Qiagen) digital PCR platform in a duplex configuration (short/long mtDNA amplicons) with an internal positive control (IPC).
Analytical validation using a NIST standard confirmed the linearity of the response for both targets over a range of at least 4–5 orders of magnitude (≈ 0.5–3 × 10³ copies/µL per reaction) and a detection limit close to a single mtDNA copy per reaction.
Analysis of forensic bone and cartilage samples revealed high concentrations of the short amplicon (up to 10⁵ copies/µL in the extract) alongside the presence of the long target, confirming the assay’s suitability for pre-selection of material for mtDNA sequencing.
Rootless hair was characterised by a significantly lower copy number and a much higher degradation ratio (short/long) than bone, which is consistent with literature reports on the variability and degradation of mtDNA in hair shafts.
Compared with published qPCR and ddPCR assays for the quantitative assessment of mtDNA, the proposed assay provides a comparable or better dynamic range, high sensitivity, and the ability to assess mtDNA degradation absolutely, making it a promising tool to support decisions regarding further analytical procedures in forensic genetics.
Authors
- Andrzej Doniec (Institute of Forensic Research, Poland)
- Andrzej Sekuła (Institute of Forensic Research, Poland)
- Marek Kowalczyk (Institute of Forensic Research, Poland)
- Miłosz Januła (Institute of Forensic Research, Poland)
- Tomasz Kupiec (Institute of Forensic Research, Poland)
Reference
[1] Kavlick, M. F. (2019). Development of a triplex mtDNA qPCR assay to assess quantification, degradation, inhibition, and amplification target copy numbers. Mitochondrion, 46, 41-50.
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