Simultaneous identification of saliva and semen by DNA methylation analysis using quantitative PCR and droplet digital PCR.

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.