Isothermal solid-phase amplification system for detection of Yersinia pestis

Area of Science:

• Molecular Biology
• Biotechnology
• Diagnostic Assays

Background:

• Conventional polymerase chain reaction (PCR) has limitations for field-based molecular diagnostics.
• Isothermal amplification methods offer alternatives to PCR for rapid, on-site testing.
• Recombinase polymerase amplification (RPA) is a sensitive and specific isothermal technique.

Purpose of the Study:

• To develop and validate an RPA-based method for the optical detection of Yersinia pestis.
• To assess the sensitivity, specificity, and speed of the developed RPA assay.
• To enable rapid identification of potential biowarfare agents in field settings.

Main Methods:

• Solid-phase RPA using immobilized thiolated forward primers on maleimide-activated microtitre plates.
• Quantitative detection via biotinylated reverse primers and streptavidin-HRP conjugate.
• Optical detection of amplified DNA at a constant temperature of 37 °C.

Main Results:

• Achieved quantitative detection of synthetic and genomic Yersinia pestis DNA in under 1 hour.
• Demonstrated high sensitivity with detection limits of 4.04*10(-13) M for ssDNA and 3.14*10(-16) M for dsDNA.
• Exhibited high specificity, with minimal response to non-target DNA sequences.

Conclusions:

• RPA is a viable isothermal amplification technique for rapid, sensitive, and specific optical detection of Yersinia pestis.
• The developed assay is suitable for field deployment in biodefense applications.
• This method provides a fast and efficient alternative to conventional PCR for pathogen detection.