Sexual assault cases are increasing in Europe and worldwide, with 1 in 3 European women reporting that they have been assaulted in adulthood, according to the Eurostat database (gender-based violence statistics). Despite growing awareness, the interpretation of the samples is hindered by late sample collection and challenges in mixture analysis. Body fluid identification (BFID) is a key component of forensic investigations, particularly in cases involving mixed biological samples such as sexual assault evidence and can act as a triage test for rapid in-sample male detection. RNA-based markers have demonstrated high specificity for BFID; however, detection and interpretation- especially in mixtures, low-template, or degraded samples- remain challenging using standard capillary electrophoresis (CE) methods.

By enabling absolute molecule quantification, digital PCR (dPCR) offers increased sensitivity for detecting trace and degraded RNA transcripts, as well as, enhanced specificity through the use of targeted probes, making it well suited for mixture analysis. Each marker can be quantified independently, facilitating clearer interpretation. While droplet-based dPCR has demonstrated the ability to detect body fluid mixtures across a range of ratios, the application of plate-based dPCR platforms in this context remains limited.

This study aims to evaluate the QuantStudio Absolute Q digital PCR system (Thermo Fisher Scientific) for the detection and identification of body fluid mixtures. RNA markers specific for vaginal fluid (MUC4) and semen (SEMG1), together with a housekeeping gene (18S rRNA), were analyzed in both single-source and mixed samples. Artificial mixtures were prepared at defined body fluid ratios (50:1 to 2:1) to simulate forensic scenarios involving major and minor contributors.
Preliminary data suggest that the QuantStudio Absolute Q system is suitable for the detection of body fluid–specific markers in forensic samples. Ongoing analyses focus on assessing specificity in single-source samples and sensitivity in mixed semen and vaginal secretions across varying ratios. The detection of minor contributors at low proportions and consistency across replicates support the robustness of the approach.

This work explores the applicability of plate-based dPCR for the analysis of body fluid mixtures in a forensic context. The platform has the potential to provide a sensitive and robust method for analyzing complex biological samples and may support the future implementation of dPCR technologies in routine forensic workflows.

Authors

• Ana Mafalda Rocha (Forensic Genetics Unit, Institute of Forensic Sciences (INCIFOR), Universidade de Santiago de Compostela, Spain; Instituto de Investigação e Inovação em Saúde da Universidade do Porto (i3S), Portugal)
• María Victoria Lareu (Forensic Genetics Unit, Institute of Forensic Sciences (INCIFOR), Universidade de Santiago de Compostela, Spain)
• Catarina Xavier (Instituto de Investigação e Inovação em Saúde da Universidade do Porto (i3S), Portugal)
• Iva Gomes (Forensic Genetics Unit, Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Portugal)