Advancing forensic body fluid identification: A comparative analysis of RT-LAMP+CRISPR-Cas12a and established mRNA-based methods

Abstract

In forensic science, the analysis of body fluid evidence determines the cellular origin of a sample, aiding in the reconstruction of a potential crime. Messenger ribonucleic acid (mRNA) based confirmatory tests address limitations of current conventional methods, providing increased specificity and sensitivity, minimal sample consumption, and the detection of a broader range of body fluids. However, they require expensive instrumentation, longer reaction times, and lack portability. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) coupled with clustered regular interspaced short palindromic repeats (CRISPR) with CRISPR-associated protein 12a (Cas12a) has the potential to overcome these challenges. This approach offers reduced testing time and cost, while potentially providing equivalent sensitivity and specificity, as observed in the field of viral diagnostics. Visual detection capabilities enable the development of rapid, portable screening tests suitable for testing at the crime scene. In the context of a sexual assault investigation, RT-LAMP+CRISPR-Cas12a could potentially increase the efficiency and detection rate. This study compares this novel method to two other mRNA-based methods, endpoint reverse transcription polymerase chain reaction (RT-PCR) multiplex assay CellTyper 2, and a real-time reverse transcription quantitative PCR (RT-qPCR) multiplex assay. The tests' sensitivity and specificity were evaluated on single-source and mixed body fluid samples, including rectal mucosa, a fluid which is minimally explored in forensic literature. The RT-qPCR assay demonstrated the highest sensitivity, specificity, and precision in mixed samples. In addition, RT-qPCR offers a greater linear dynamic range, faster processing time and easier methodology compared to CellTyper 2, only limited by its expensive nature. Notably, rectal mucosa samples exhibited non-specific marker expression of CellTyper 2 markers and expression of CYP2B7P (vaginal fluid) for all methods. This emphasises the need for a dedicated rectal mucosa marker. RT-LAMP+CRISPR-Cas12a exhibited a high specificity, displaying off-target expression of CYP2B7P in two fluid types. However, the method lacked sensitivity and precision for most markers except MMP3 (menstrual blood), demonstrating detection down to 1:10,000 with 100 % specificity. RT-LAMP+CRISPR requires further development, but its quick, inexpensive nature and high specificity suggest it has potential as a confirmatory test that could reduce the limitations of existing methods.

Keywords: Endpoint RT-PCR; Forensic mRNA body fluid identification; RT-LAMP+ CRISPR-Cas12a; Real-time RT-qPCR; Rectal mucosa.