Multiplex detection of the big five carbapenemase genes using solid-phase recombinase polymerase amplification

Area of Science:

• Molecular Biology
• Antimicrobial Resistance
• Diagnostic Development

Background:

• The

Purpose of the Study:

• To develop a rapid molecular detection method for the "big five" carbapenemase genes.
• To utilize isothermal amplification via recombinase polymerase amplification (RPA) for enhanced sensitivity and specificity.
• To establish a streamlined assay for potential point-of-care application.

Main Methods:

• Development of a spatially separated multiplex recombinase polymerase amplification (RPA) assay.
• Utilisation of tailed oligonucleotides for hybridisation and a solid-phase RPA format with immobilised primers.
• Integration of horseradish peroxidase reporter probe with both microplate reader and portable Raman spectrometer detection.

Main Results:

• Successful detection of all five "big five" carbapenemase genes with femtomolar limits of detection.
• Demonstrated up to a 19-fold enhancement in detection limit using a portable Raman spectrometer.
• Achieved a 2-hour assay time, including a 40-minute RPA amplification step at 37°C.

Conclusions:

• The developed solid-phase RPA assay is the first to detect the "big five" carbapenemase genes.
• The assay demonstrates high sensitivity, reduced complexity, and potential for cost-effectiveness.
• This represents a significant milestone towards automated point-of-care diagnostics for carbapenemase resistance.