Innovation
Focusing on the research and development and application of isothermal nucleic acid amplification technology
Development of a Rapid Fluorescent RAA Assay for the Detection of Pasteurella multocida—Du Qiuming
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To establish a sensitive, specific, simple, and rapid method for detecting Pasteurella multocida, we designed specific primers and probes based on the conserved sequence of the kmt1 gene in P. multocida. After optimizing and screening reaction conditions, we developed a recombinase-mediated isothermal amplification (RAA) fluorescence detection method. The results showed that the established RAA fluorescence assay for P. multocida could specifically detect the bacterium within 20 minutes at a constant temperature of 39°C. The assay exhibited no cross-reactivity with other pathogens, including Riemerella anatipestifer, Haemophilus parasuis, Mycoplasma, Streptococcus, Escherichia coli, Eperythrozoon, and Neospora caninum. The method’s limit of detection was as low as 10 copies/μL, and the coefficients of variation in both intra-assay and inter-assay repeatability tests were less than 10%. When applied to the analysis of 45 clinical samples, the assay yielded a positive rate of 33.33%, which was consistent with the results obtained by real-time fluorescent quantitative PCR. The RAA fluorescence assay for P. multocida developed in this study is simple to perform, has a short reaction time, and demonstrates high specificity and sensitivity. Moreover, it does not rely on complex instrumentation, making it well-suited for rapid detection of P. multocida in clinical settings.
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