Multiplex methylation-specific PCR for distinguishing semen, saliva, and blood

Analysis of body fluids obtained from crime scenes provides important information for directing criminal investigations. Generally, chemical and immunological techniques, along with molecular methods that utilize RNA, are used for body fluid identification. However, these methods have some limitations, such as handling difficulty, sample consumption, false-positive or -negative results, and sample instability. To address these problems, several researchers have utilized DNA methylation for body fluid identification, including microarrays, combined bisulfite restriction analysis, single-base extension, and pyrosequencing. Although these methods have the advantage of simultaneously analyzing multiple target sites at the same time, they require considerable time and cost. In this study, a methylation-specific PCR (MSP)-based method was used to simultaneously discriminate between three human body fluids (semen, saliva, and blood). We designed primers with different amplicon sizes for each specific CpG region in the three body fluids (ASIC4 for semen, FAM43A for saliva, and FOXO3 for blood). For the accurate analysis, methylation and unmethylation primers were used separately for different amplifications. This MSP-based method can discriminate body fluids in unknown samples, although caution is required when analyzing mixed or low-quantity DNA samples. Our multiplex MSP-based approach enables confirmation of human body fluids and may provide a potentially simpler workflow compatible with conventional capillary electrophoresis platforms compared with methods such as the SNaPshot assay. We anticipate that this multiplex PCR system will be helpful in criminal investigations.