Forensic human identification by STR profiling via capillary electrophoresis (CE) remains the gold standard, yet there are still limitations with low-template DNA, degraded samples, and inhibitor-rich crime scene matrices. Allele dropout and partial profiles at inputs below 100 pg can prevent statistically actionable random match probability (RMP), while common crime scene compounds such as hematin and humic acid may cause amplification failure or significant profile imbalance even at standard DNA inputs. In practice, these challenges arise regularly in forensic casework and remain a modern analytical challenge

We present HyperMatch, a 91-plex assay developed using Hyperplex PCR (hpPCR). hpPCR integrates multiplex PCR with padlock probe-mediated rolling circle amplification (RCA) and digital fluorescence imaging-based counting of individual RCA products labelled with nanoparticle probes. By converting sub-picomolar PCR amplicon concentrations into discrete, optically countable RCA products, hpPCR maintains high analytical sensitivity even when upstream amplification is severely compromised, providing an intrinsic and mechanistic resistance to inhibition that is distinct from mastermix-level optimisation alone. Compatibility with standard off-the-shelf PCR mastermixes further extends this inhibition tolerance and simplifies implementation.

HyperMatch targets 44 biallelic insertion/deletion polymorphisms (indels) with allele frequencies of approximately 0.5 across all major human populations, yielding a theoretical RMP of approximately 2.5 × 10⁻²⁰. To maximize the probability of genotyping success from degraded biological material, all target amplicons were kept deliberately short, with lengths ranging from 100 to 168 bp. The assay was optimized and stress-tested using five commercially available single-source DNA reference samples, with panel performance evaluated across four analytically critical parameters: (1) sensitivity at low total DNA input; (2) sensitivity for the detection of a minor DNA contributor in a binary mixture; (3) resistance to inhibition, using hematin and humic acid as representative PCR inhibitor models; and (4) tolerance to DNA degradation. HyperMatch achieved an RMP of 6 × 10⁻¹⁴ from a DNA input of 10 pg, detected a minor contributor at a 1% fraction in a binary mixture, and retained an RMP of at least 1 × 10⁻⁹ in the presence of 1 mM hematin or 400 µg/mL humic acid at 1 ng DNA input, as well as from 1 ng of DNA subjected to degradation through 70 minutes of heat treatment at 99°C. These results position HyperMatch as a promising tool concerning sensitivity, mixture resolution, and PCR inhibitor resistance for forensic genotyping.

Authors

• Danai Nikou (Aplex Bio, Stockholm, Sweden)
• Ruben R. G. Soares (Aplex Bio AB, Sweden)
• Johannes Hedman (National Forensic Centre, Swedish Police Authority, Sweden)
• Maja Sidstedt (National Forensic Centre, Swedish Police Authority, Sweden)
• Margarita Psallida (Aplex Bio AB, Sweden)
• Ciara Morrison (Aplex Bio AB, Sweden)
• Eleni Psaroudaki (Aplex Bio AB, Sweden)
• Umear Naseem (Aplex Bio AB, Sweden)
• Erik Larsson (Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, The Sahlgrenska Academy, University of Gothenburg, Sweden)
• Markus Lindberg (Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, The Sahlgrenska Academy, University of Gothenburg, Sweden)
• Alice Schiller (Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, The Sahlgrenska Academy, University of Gothenburg, Sweden)
• Samuel Narmack (Aplex Bio, Stockholm, Sweden)