Validation of a novel fluorescent probe-based real-time PCR assay to detect saliva-specific unmethylated CpG sites for saliva identification

The identification of saliva from forensic samples is often important to establish what happened at a crime scene, especially in sexual assault cases. Recently, CpG sites that are specifically methylated or unmethylated in saliva have been reported as markers for saliva identification. In this study, we designed a fluorescent probe-based real-time polymerase chain reaction (PCR) assay for analyzing the methylation status of two neighboring CpG sites, which we previously found were saliva-specifically unmethylated. Specificity analysis using various types of body fluid/tissue samples demonstrated a probe detecting the unmethylation of the two CpG sites reacted only to saliva DNA, indicating this probe as an all-or-nothing marker for the presence of saliva DNA. Sensitivity analysis demonstrated that the detection limit was 0.5 ng saliva DNA as input for bisulfite conversion, while we confirmed a negative effect of larger amounts of non-saliva DNA on sensitivity in the analysis of saliva-vaginal DNA mixtures. We finally validated the applicability of this test to swabs from licked skin and bottles after drinking as mock forensic samples in comparison with other saliva-specific markers. We confirmed the potential usefulness of this test for skin samples, from which a saliva-specific mRNA was not detected reliably, while the ingredients in several beverages might affect methylation analysis. Given the simplicity of real-time PCR as well as the high specificity and sensitivity of the test, we believe the developed method is suitable for routine forensic analysis and can play an important role in saliva identification.