Introduction

Biological evidence detected at crime scenes plays an important role in case investigations [1, 2]. Body fluid identification aids in reconstructing the sequence of events at crime scenes. Generally, tests for discriminating human body fluids include chemical, enzymatic, or immunological assays. For example, luminol and leucomalachite green are chemical assays used for blood detection [3]. Phadebas® and SALIgAE® are also chemical tests to detect saliva in forensics [4]. Seminal acid phosphatase and prostate-specific antigen tests are useful chemical and immunological methods for semen detection [5]. These assays facilitate the straightforward identification of body fluids; however, some tests have several limitations such as sample consumption and their low sensitivity that can lead to false-positive or -negative results. Other assays that leverage differentially expressed mRNA levels between tissues have also been developed. However, they have limitations attributed to the greater instability of RNA compared with that of DNA and the need for careful handling to prevent degradation by ubiquitous RNases [6–10]. To address these issues, investigators have explored more accurate body fluid identification methods by leveraging epigenetics [11–13].

Epigenetics refers to various structural changes in a genome without alterations in the DNA sequence. Epigenetic modifications include histone modifications, DNA methylation, noncoding RNA action, and chromatin remodeling [14]. Among them, DNA methylation, a process in which a methyl group is added to the 5′ carbon of the cytosine within the pyrimidine base, regulates gene expression. Most methylation occurs at CpG sites, with 70–80% of these sites methylated in the human genome [2, 14–18]. DNA methylation controls mammalian development, including cell differentiation, embryonic development, X chromosome inactivation, genomic imprinting, and the inactivation of tumor suppressor genes [19, 20].

The identification of body fluids can be achieved by analyzing tissue-specific differentially methylated regions [21, 22]. The methods for analyzing methylation are diverse and include bead-based microarrays, combined bisulfite restriction analysis, single-base extension, pyrosequencing, and methylation-specific PCR (MSP). Herman et al. (1996) devised MSP, a simple and powerful method to confirm a specific methylation region in bisulfite-converted DNA using PCR [23]. Bisulfite treatment converts unmethylated cytosines to uracil, whereas methylated cytosines remain unchanged. During PCR, uracils are amplified to thymine, allowing the distinction between methylated and unmethylated cytosines. The MSP method requires methylated (M) and unmethylated (U) primers targeting specific CpG sites, and PCR amplification products are analyzed using gel electrophoresis to determine methylation status. Additionally, samples can be sensitively analyzed using capillary electrophoresis (CE) by attaching fluorescent tags to primers. Because of its simplicity, MSP is widely used to diagnose diseases associated with DNA methylation [24–26].

In this study, we distinguished three human body fluids: semen, saliva, and blood, by applying a multiplex MSP system. We designed M and U primers targeting the specific hypermethylated CpG sites in each body fluid sample (ASIC4 for semen, FAM43A for saliva, and FOXO3 for blood) based on the previous published papers [27, 28]. The sizes of the amplicons differed for each body fluid. Successful identification of all three body fluids in a single reaction was accomplished using case and mixed test samples. Furthermore, data analysis using CE instruments offers a more convenient, straightforward, and expedited alternative to traditional approaches.