Development of mRNA-based body fluid identification using reverse transcription loop-mediated isothermal...

Area of Science:

• Forensic Science
• Molecular Biology
• Biochemistry

Background:

• Accurate identification of body fluids in forensic samples is crucial for criminal investigations.
• Messenger RNA (mRNA)-based methods and reverse transcription polymerase chain reaction (RT-PCR) have been utilized for body fluid identification.
• There is a need for simple, rapid, and sensitive assays for forensic body fluid analysis.

Purpose of the Study:

• To develop and evaluate reverse transcription loop-mediated isothermal amplification (RT-LAMP) as a simple and rapid assay for identifying blood, semen, and saliva.
• To assess the specificity and sensitivity of the developed RT-LAMP assays.
• To determine the applicability of RT-LAMP assays to forensic casework samples.

Main Methods:

• Selected marker genes: Hemoglobin beta (HBB) for blood, transglutaminase 4 (TGM4) for semen, and statherin (STATH) for saliva.
• Developed a single-step RT-LAMP assay combining reverse transcription and DNA amplification under isothermal conditions (60 min).
• Evaluated assay specificity by testing against five forensically relevant body fluids and assessed sensitivity through detection limits.

Main Results:

• RT-LAMP assays successfully identified blood, semen, and saliva with marker-specific amplification.
• No cross-reactivity was observed among the tested body fluids, indicating high specificity.
• Detection limits were 0.3 nL for blood, 30 nL for semen, and 0.3 μL for saliva, with sensitivity comparable to RT-PCR.
• The RT-LAMP assays proved applicable to real forensic casework samples.

Conclusions:

• RT-LAMP is a simple, rapid, and sensitive method for identifying common forensic body fluids.
• The assay's performance is comparable to established methods like RT-PCR.
• RT-LAMP is a valuable tool for body fluid identification in forensic casework.