Rapid and inexpensive body fluid identification by RNA profiling-based multiplex High Resolution Melt (HRM) analysis

Area of Science:

• Forensic Science
• Molecular Biology
• Biochemistry

Background:

• Accurate identification of biological material on evidence is critical for criminal investigations.
• Existing protein-based methods and molecular techniques like capillary electrophoresis (CE) and quantitative RT-PCR (qRT-PCR) have limitations in identifying all body fluids and tissues.
• Current mRNA profiling assays are costly due to fluorescently labeled reagents and can be time-consuming, with limited multiplexing capabilities.

Purpose of the Study:

• To develop simplified, cost-effective, and rapid messenger RNA (mRNA) profiling assays for identifying forensically relevant biological fluids and tissues.
• To overcome the limitations of existing CE and qRT-PCR methods in terms of cost, time, and multiplexing capacity.
• To introduce High Resolution Melt (HRM) assays for simultaneous detection of multiple biomarkers.

Main Methods:

• Development of singleplex and multiplex High Resolution Melt (HRM) assays utilizing unlabeled PCR primers and a single saturating intercalating fluorescent dye (Eva Green).
• Incorporation of specific biomarkers for various body fluids: IL19 (vaginal secretions), IL1F7 (skin), ALAS2 (blood), MMP10 (menstrual blood), HTN3 (saliva), and TGM4 (semen).
• Unique design of duplex and triplex HRM assays for simultaneous detection of multiple targets within a single reaction.

Main Results:

• The developed HRM assays successfully identified specific body fluids and tissues using distinct melt peaks.
• These assays require only unlabeled PCR primers and a single fluorescent dye, significantly reducing reagent costs.
• Duplex and triplex HRM assays demonstrated the capability for simultaneous detection of multiple targets, enhancing efficiency.
• Initial performance evaluation indicates the potential for rapid and cost-effective screening of biological evidence.

Conclusions:

• High Resolution Melt (HRM) assays offer a promising alternative for the identification of forensically relevant biological fluids and tissues.
• The developed HRM assays provide a rapid, cost-effective, and simplified approach to biological evidence screening.
• This molecular-based strategy addresses the limitations of traditional methods, improving forensic analysis capabilities.