Identifying tissue source types of human peripheral blood, menstrual blood, vaginal secretion, semen, and saliva left at a crime scene can offer crucial clues for case investigation. Although messenger RNA (mRNA) profiling has become a robust tool for body fluid identification, conventional methods remain constrained by time-consuming workflows and dependence on laboratory infrastructure, hindering rapid on-site forensic analysis. To address these limitations, this study designed the RPA-CRISPR-Cas12 experiment and independently developed an RPA-CRISPR-Cas12a-LFD detection system, which achieved simple, rapid (<40 min), and accurate forensic identification of five kinds of critical body fluids. Initially, we mined the Genotype-Tissue Expression database and related literatures to screen candidate mRNA markers, subsequently employed RT-qPCR to evaluate their expression levels, and applied the Boruta algorithm to optimize the number of mRNA markers, ultimately identifying five mRNA markers: HBB (peripheral blood), MMP10 (menstrual blood), DKK4 (vaginal secretion), AKAP4 (semen), and HTN3 (saliva). The developed system exhibited exceptional specificity. Furthermore, the sensitivity values of HBB, MMP10, DKK4, AKAP4, and HTN3 markers in RPA-CRISPR-Cas12 and RPA-CRISPR-Cas12a-LFD detection systems were 0.1, 0.1, 1, 0.1, 0.1 ng; and 0.1, 0.1, 1, 1 and 1 ng, respectively. Among them, the sensitivity of HBB and DKK4 markers was superior to that of previous studies. Notably, the platform successfully resolved mixed samples (excluding AKAP4 marker), and MMP10 and DKK4 markers retained detectability in aged menstrual blood and vaginal secretion samples. The proposed method provides the groundwork for further development of tissue origin identification tools of forensic body fluids, which may eventually be suitable for on-site rapid detection environments.
CRISPR Gene Editing