Concluding remarks

Analysis of methylation at specific CpG sites can be useful for body fluid prediction in forensic studies. While several analytical methods, such as SNaPshot, chemical, and RNA-based assays, have been developed for body-fluid identification, some approaches may be associated with limitations, including the potential for false-positive or false-negative results, sample consumption, and the inherent instability of RNA. Therefore, we devised multiplex PCR primer sets that could simultaneously detect three types of body fluids (semen, saliva, and blood) using methylation-specific PCR. Three markers (ASIC4, FAM43A, and FOXO3), which have high β-scores for detecting specific body fluids, were selected based on previous research papers by Lee et al. [27] and Park et al. [28]. The methylation primers targeted only one type of body fluid; however, low RFU values of non-target markers were detected in all samples amplified with M primers. This phenomenon may be attributed to the β-scores for each body fluid. The methylation patterns were correctly represented in the mixture and control tests. Analyzing low amounts of DNA is challenging; however, the MSP method employed in this study provided reliable results even with limited DNA input, as evidenced by the serial dilution experiments. Furthermore, the MSP assay successfully identified the types of biological fluid in all mixed samples, demonstrating its potential to provide valuable investigative information regarding the circumstances of evidence deposition. In validation tests using evidentiary samples, the MSP primer set demonstrated the ability to discriminate potential sources of biological material, suggesting its potential applicability in forensic casework. The ability to detect and differentiate multiple biological fluids within a single-sample offers enhanced discriminatory power for forensic investigations, particularly when conventional methods yield ambiguous results. Nonetheless, forensic samples obtained from actual crime scenes may often contain substantially lower amounts of DNA than the inputs tested in this study. Therefore, further research is needed to develop more sensitive methods that can identify body fluids using minimal DNA. Furthermore, we aim to develop primer sets capable of distinguishing vaginal fluid and menstrual blood in sexual assault cases. This cutoff (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\overline{x}}{(neg)}+{{k}{m}\cdot SD}_{(neg)}$$\end{document}) was used as a conservative upper threshold to reduce false-positive calls by excluding the upper tail of the negative distribution, when specificity is prioritized in forensic decision-making.