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

Area of Science:

• Forensic Science
• Molecular Biology
• Biochemistry

Background:

• Current body fluid analysis in forensics faces limitations in specificity, sensitivity, and sample consumption.
• Messenger ribonucleic acid (mRNA) based tests offer improved accuracy but require expensive equipment and time.
• Novel methods are needed to enhance forensic body fluid identification, especially for challenging samples like rectal mucosa.

Purpose of the Study:

• To evaluate the performance of a novel RT-LAMP+CRISPR-Cas12a method for body fluid identification.
• To compare RT-LAMP+CRISPR-Cas12a against established mRNA-based methods: CellTyper 2 (RT-PCR) and RT-qPCR.
• To assess the sensitivity and specificity of these methods on single-source and mixed body fluid samples, including rectal mucosa.

Main Methods:

• Real-time reverse transcription quantitative PCR (RT-qPCR) multiplex assay.
• Endpoint reverse transcription polymerase chain reaction (RT-PCR) multiplex assay (CellTyper 2).
• Reverse-transcription loop-mediated isothermal amplification coupled with CRISPR-Cas12a (RT-LAMP+CRISPR-Cas12a).

Main Results:

• RT-qPCR demonstrated the highest sensitivity, specificity, and precision in mixed samples.
• RT-LAMP+CRISPR-Cas12a showed high specificity but lacked sensitivity for most markers, except MMP3 in menstrual blood.
• Rectal mucosa samples presented challenges, including non-specific marker expression and the need for dedicated markers.

Conclusions:

• RT-qPCR is a highly sensitive and precise method for forensic body fluid analysis, despite its cost.
• RT-LAMP+CRISPR-Cas12a holds potential as a rapid, inexpensive, and specific confirmatory test, requiring further optimization.
• Further research is needed to develop specific markers for challenging forensic samples like rectal mucosa.