Simultaneous identification of saliva and semen by DNA methylation analysis using quantitative PCR and droplet digital PCR.
TL;DR
It is demonstrated that ddPCR has the potential for application in forensic science as it effectively identified saliva in mixtures up to a 1:50 ratio and semen up to a 200:1 ratio.
Abstract
DNA methylation analysis is crucial in forensic science for age estimation, body fluid identification, and differentiating monozygotic twins. While massively parallel sequencing (MPS) and single-base extension (SBE) are standard, they are often time-consuming and costly. This study evaluated the feasibility of using quantitative PCR (qPCR) and droplet digital PCR (ddPCR) to identify saliva and semen. DNA was bisulfite-converted using an EZ DNA Methylation-Gold Kit and two body-fluid-specific cytosine-phosphate-guanine (CpG) markers (cg09652652-2d for saliva and cg17610929 for semen) were analyzed using a QX600 droplet digital PCR system. This assay showed high sensitivity for samples with concentrations greater than 5 copies/μL. In contrast, qPCR yielded Ct values above 30 for the same samples. To assess specificity, blood, menstrual blood, vaginal secretion, and nasal secretion samples were tested for cross-reactivity with saliva and semen. The saliva-specific marker was amplified in saliva samples and slight amplification was also observed in nasal secretion samples. The semen-specific marker was detected only in semen samples. ddPCR effectively identified saliva in mixtures up to a 1:50 ratio and semen up to a 200:1 ratio. Moreover, ddPCR demonstrated greater tolerance to PCR inhibition by humic acid compared to qPCR. The DNA degradation test showed no significant difference between degraded and non-degraded saliva samples (paired t-test, p > 0.05). In contrast, a statistically significant difference was observed in semen. These findings demonstrate that ddPCR has the potential for application in forensic science.