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Updated: Oct 2, 2026
Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control
Published on: March 30, 2015
Development and evaluation of a recombinase-aided amplification-lateral flow dipstick assay for rapid and sensitive
Shu Wang1, Yingkai Zhu1, Yuxin Wang1
1College of Animal Science and Technology, Shihezi University, Shihezi, Xinjiang, China.
Background:
Mycoplasma synoviae (MS) is a critical avian pathogen responsible for enormous economic losses in global poultry production. Timely prevention and control of MS epidemics rely heavily on rapid, sensitive and accurate diagnostic approaches, while conventional detection methods remain time-consuming and complicated. Therefore, we developed a visual on-site detection system based on recombinase-aided amplification combined with lateral flow dipstick for MS (RAA-LFD-MS assay).
Methods:
According to the design rules for RAA primers and exo-probes, specific primers and a probe were designed targeting the conserved region of the MS vlhA gene. A recombinant plasmid containing the target fragment was constructed as the positive standard. After primer-probe screening, key reaction parameters including primer concentration, probe concentration, reaction temperature, and incubation time were systematically optimized. The analytical specificity, sensitivity, and repeatability of the established assay were fully evaluated. To further evaluate the clinical performance of our RAA-LFD-MS assay, a total of 100 clinical specimens suspected of MS infection were tested in parallel using this newly established method, conventional polymerase chain reaction (PCR), and quantitative real-time PCR (qPCR).
Results:
The RAA reaction was completed within 20 min at a constant temperature of 37 °C, and amplification products were visually read within 3-5 min using a lateral flow dipstick without sophisticated equipment. The total detection time, including the DNA extraction step, is completed within 40 min. Methodological validation revealed that the RAA-LFD assay exhibited high specificity, with no cross-reactivity observed against other common avian pathogens. The assay also showed satisfactory repeatability and high sensitivity, with a limit of detection of 2.63×100 copies/μL. In the clinical sample evaluation, the coincidence rate between RAA-LFD and PCR was 96%, while the agreement between RAA-LFD and qPCR reached 100%.
Conclusion:
In summary, the established RAA-LFD assay is rapid, user-friendly, highly specific, and sensitive. To further improve the RAA-LFD-MS assay for routine clinical and field use, future efforts should prioritize the optimization of rapid DNA extraction, accessible temperature-control devices, and reagent lyophilization.
