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Focusing on the research and development and application of isothermal nucleic acid amplification technology
多杀性巴氏杆菌荧光RAA快速检测方法的建立-杜秋明
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To establish a sensitive, specific, simple, and rapid detection method for *Pasteurella multocida*, specific primers and probes were designed based on the conserved sequence region of the *kmt1* gene in *P. multocida*. Through optimization and screening of reaction conditions, a recombinase-aided amplification (RAA) fluorescence assay was successfully developed. The results demonstrated that the established RAA fluorescence assay can specifically detect *P. multocida* within 20 minutes at a constant temperature of 39°C, with no cross-reactivity observed against *Riemerella anatipestifer*, *Haemophilus parasuis*, Mycoplasma species, Streptococcus species, Escherichia coli, Eperythrozoon species, or Neospora caninum. The method achieved a detection limit as low as 10 copies/μL, and both intra- and inter-assay repeatability tests showed coefficients of variation below 10%. When applied to 45 clinical samples, the assay yielded a positive rate of 33.33%, which was consistent with the results obtained by fluorescent quantitative PCR. This study has successfully developed a fluorescent RAA assay for *P. multocida* that is easy to perform, requires minimal time, exhibits high specificity and sensitivity, and does not rely on complex equipment—making it well-suited for rapid clinical detection of *P. multocida*.
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