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Updated: Jul 4, 2026
Optimized PCR-based Detection of Mycoplasma
Published on: June 20, 2011
Rapid and visual detection of Mycoplasma synoviae by recombinase-aided amplification assay combined with a lateral
Wenlong Xia1, Ke Chen1, Wensong Liu1
1Yancheng Engineering Research Center of Animal Biologics, College of Marine and Biological Engineering, Yancheng Teachers University, Yancheng 224002, China.
Abstract:
Mycoplasma synoviae (MS) is an important avian pathogen that has brought substantial economic losses to the global poultry industry. Fast and accurate diagnosis is one of the critical factors for the control of MS infection. This study established a simple, rapid and visual detection method for MS using a recombinase-aided amplification (RAA) combined with a lateral flow dipstick (LFD). The reaction temperature and time of the RAA-LFD assay were optimized after selecting the primers and probe, and the specificity and sensitivity rates were analyzed. The results showed that RAA could amplify the target gene in 20 min at a constant temperature of 38°C, and the amplification products could be visualized by LFD within 5 min. There was no cross-reaction with Mycoplasma gallisepticum (MG), Pasteurella multocida (P. multocida), Escherichia coli (E. coli), Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), and avian reovirus (ARV). Furthermore, the RAA-LFD assay exhibited high sensitivity with a detection limit of 10 copies/μL. A total of 128 clinical samples with suspected infection of MS were tested by RAA-LFD, PCR, and real-time fluorescence quantitative PCR (RFQ-PCR). The coincidence rate of the detection results was 95.3% between RAA-LFD and PCR, and 98.4% between RAA-LFD and RFQ-PCR. These results suggested that the RAA-LFD method established in the present study was easy to use and was associated with strong specificity and high sensitivity. This method was very suitable for the rapid detection of MS in clinical practice.
Insights
A new recombinase-aided amplification combined with lateral flow dipstick (RAA-LFD) assay provides rapid, visual detection of Mycoplasma synoviae (MS) in poultry. This highly specific and sensitive method is suitable for quick diagnosis in clinical settings.
Area of Science:
- Avian Pathology
- Molecular Diagnostics
- Veterinary Microbiology
Background:
- Mycoplasma synoviae (MS) causes significant economic losses in the poultry industry.
- Rapid and accurate diagnosis is crucial for effective MS infection control.
- Existing diagnostic methods may lack speed or simplicity for field application.
Purpose of the Study:
- To develop a simple, rapid, and visual detection method for Mycoplasma synoviae (MS).
- To establish a diagnostic tool combining recombinase-aided amplification (RAA) with a lateral flow dipstick (LFD).
Main Methods:
- Optimization of RAA reaction conditions (temperature, time) and primer/probe selection.
- Assessment of assay specificity against common avian pathogens.
- Determination of assay sensitivity and validation using clinical samples compared to PCR and RFQ-PCR.
Main Results:
- The RAA-LFD assay successfully amplified the target gene in 20 minutes at 38°C, with results visualized by LFD in 5 minutes.
- The assay demonstrated high specificity, showing no cross-reactivity with Mycoplasma gallisepticum and other tested pathogens.
- Sensitivity was high (10 copies/μL), and clinical sample concordance was 95.3% with PCR and 98.4% with RFQ-PCR.
Conclusions:
- The developed RAA-LFD method is a user-friendly, highly specific, and sensitive tool for MS detection.
- This assay is well-suited for rapid diagnosis of Mycoplasma synoviae in clinical veterinary practice.
- The RAA-LFD assay offers a valuable alternative for timely MS infection management in poultry.
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