Research Note: Real-time fluorescence-based recombinase-aided amplification for rapid detection of Mycoplasma...

Area of Science:

• Veterinary Microbiology
• Molecular Diagnostics
• Poultry Health

Background:

• Mycoplasma synoviae (MS) is a significant poultry pathogen globally.
• Current MS detection methods are complex, time-consuming, and require specialized equipment.
• There is a critical need for rapid and simple diagnostic tools for MS.

Purpose of the Study:

• To develop and validate a novel real-time fluorescence-based recombinase-aided amplification (RF-RAA) technique for MS nucleic acid detection.
• To establish a rapid, sensitive, and specific method for diagnosing MS infection in poultry.
• To provide a visually interpretable diagnostic tool for resource-limited settings.

Main Methods:

• Development of a real-time fluorescence-based recombinase-aided amplification (RF-RAA) assay.
• Amplification of target MS nucleic acids within 20 minutes at 39°C.
• Validation using real-time fluorescence monitoring and portable blue light transillumination for visual inspection.
• Testing on 71 clinical chicken throat swab samples.

Main Results:

• The RF-RAA method achieved target gene amplification in under 20 minutes.
• High specificity was demonstrated, with no cross-reactivity against common poultry pathogens.
• The assay showed high sensitivity, detecting as low as 10 copies/μL of standard plasmid.
• Diagnostic coincidence with real-time quantitative polymerase chain reaction (qPCR) was 100% for fluorescence monitoring and 97.2% for visual observation.

Conclusions:

• The developed RF-RAA method is a rapid and visually observable approach for MS detection.
• This technique offers a novel diagnostic tool for MS infection, particularly beneficial in resource-limited environments.
• RF-RAA provides a valuable alternative to conventional laboratory methods for poultry disease surveillance.