Human DNA confirmation and body fluid identification are essential preliminary steps in forensic casework, directly determining the probative value of biological evidence for criminal investigations. Conventional methods such as qPCR and capillary electrophoresis are constrained by long detection cycles, reliance on thermal cycling equipment, and poor adaptability to on-site rapid analysis. For trace, degraded, or environmentally compromised crime scene samples, there is an urgent need for a rapid, sensitive, and specific assay. This study aimed to develop a one-tube isothermal method integrating reverse transcription recombinase polymerase amplification (RT-RPA) and the CRISPR-Cas12a system for simultaneous human DNA confirmation and body fluid identification in a single reaction.

The human-specific mitochondrial cytochrome b (Cytb) gene was selected as the target for human DNA identification. Five body fluid-specific mRNA markers were curated from the Genotype-Tissue Expression (GTEx) database: ALAS2 (peripheral blood), STC1 (menstrual blood), PRM1 (semen), SERPINB3 (vaginal secret), and MUC7 (saliva). Specific RT-RPA primers and matched crRNAs were designed for each target, and a one-tube multiplex system was optimized to integrate reverse transcription, isothermal amplification, and CRISPR detection, eliminating aerosol contamination. Assay specificity was validated via cross-reactivity testing using 68 samples from five body fluids and seven common non-human species (mouse, rabbit, pig, chicken, dog, cat). Sensitivity was assessed via serially diluted total RNA, and practical performance was verified against RT-qPCR.

The assay exhibited excellent specificity: Cytb only shows fluorescence signals in human samples, with no cross-reaction with non-human species. Each body fluid marker showed target-specific signals, with no cross-reactivity between fluid types except for expected weak reactivity of the vaginal secretion marker in some menstrual blood samples. The limit of detection (LOD) was 10 pg for Cytb and 100 pg for mRNA markers. The assay successfully detected target signals in simulated forensic samples, including those stored at room temperature for 6 months and samples mixed with non-human DNA. Results were concordant with RT-qPCR, and the assay demonstrated superior sensitivity for degraded or low-template samples.

This study established a novel RT-RPA/CRISPR-Cas12a isothermal assay for simultaneous human DNA confirmation and five body fluids typing in a single reaction. Aligned with ISFG’s focus on innovative forensic biology technologies, the method offers high specificity and sensitivity, and completes within 50 minutes without thermal cycling equipment. It is applicable for both routine laboratory analysis and on-site crime scene detection, improving the efficiency of forensic biological evidence examination.

Authors

• Hewen Yao (Department of Forensic Genetics, West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, China)
• Chaoran Sun (Department of Forensic Genetics, West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, China)
• Feng Song (Department of Forensic Genetics, West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, China)
• Haibo Luo (Department of Forensic Genetics, West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, China)