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Rapid and sensitive detection of Mycoplasma synoviae using RPA combined with Pyrococcus furiosus Argonaute
Yanli Zhao1, Yuhua Zhang1, Weiqing Wu1
1Guangdong Key Laboratory for Veterinary Pharmaceutics Development and Safety evaluation, College of Veterinary Medicine, South China Agricultural University, Guangzhou, 510642, China; Guangdong Laboratory for Lingnan Modern Agriculture, South China Agricultural University, Guangzhou, 510642, China.
Abstract:
Mycoplasma synoviae (MS) is an important pathogen in laying hens and causes serious economic losses in poultry production. Rapid, accurate and specific detection is important for the prevention and control of MS. Argonaute from Pyrococcus furiosus (PfAgo) is emerging as a nucleic acid detector that works via "dual-step" sequence-specific cleavage. In this study, an MS detection method combining recombinase polymerase amplification (RPA) and PfAgo was established. Through elaborate design and screening of RPA primers and PfAgo gDNA and condition optimization, amplification and detection procedures can be completed within 40 min, whereas the results were superficially interpreted under UV and blue light. The sensitivity for MS detection was 2 copies/µL, and the specificity results showed no cross reaction with other pathogens. For the detection of 31 clinical samples, the results of this method and qPCR were completely consistent. This method provides a reliable and convenient method for the on-site detection of MS that is easy to operate without complex instruments and equipment.
Insights
A new method using recombinase polymerase amplification (RPA) and Pyrococcus furiosus Argonaute (PfAgo) allows rapid, sensitive, and specific detection of Mycoplasma synoviae in poultry. This technique offers a convenient tool for on-site diagnostics.
Area of Science:
- Veterinary Microbiology
- Molecular Diagnostics
- Biotechnology
Background:
- Mycoplasma synoviae (MS) significantly impacts poultry health and economic viability.
- Accurate and rapid detection of MS is crucial for effective disease management.
- Existing diagnostic methods may require complex equipment or longer turnaround times.
Purpose of the Study:
- To develop a rapid, sensitive, and specific diagnostic assay for Mycoplasma synoviae.
- To integrate recombinase polymerase amplification (RPA) with Pyrococcus furiosus Argonaute (PfAgo) for nucleic acid detection.
- To establish a convenient and accessible method for on-site MS detection.
Main Methods:
- Development and optimization of a combined RPA-PfAgo assay for MS detection.
- Screening of RPA primers and PfAgo conditions for efficient amplification and cleavage.
- Validation of the assay's sensitivity, specificity, and performance on clinical samples.
Main Results:
- The assay achieved a detection limit of 2 copies/µL for MS.
- High specificity was confirmed, with no cross-reactivity observed against other relevant pathogens.
- The RPA-PfAgo method demonstrated complete concordance with quantitative PCR (qPCR) on 31 clinical samples.
- Assay completion time was under 40 minutes with simple visual interpretation.
Conclusions:
- The developed RPA-PfAgo assay is a reliable and user-friendly tool for rapid on-site detection of Mycoplasma synoviae.
- This method eliminates the need for complex instrumentation, facilitating its use in diverse settings.
- The assay offers a valuable alternative for timely MS diagnosis in poultry production.
