Abstract: In order to establish a recombinase polymerase amplification (RPA) diagnostic method for Pasteurella multocida from goat, the pET-28a(+)-KMT1 plasmid standard was constructed based on the KMT1 gene sequence of Pasteurella multocida from goat. The specific primers and RPA fluorescent probe based on RPA technology were designed to establish the optimal reaction system of Real-time RPA. The sensitivity of the method was detected and the correlation curve was drawn by 10 fold gradient diluted plasmid standard. The genomic DNA of 10 different strains were used as templates to verify the specificity of the method. Finally, the reliability of the method was verified by the tissue samples of goats and mice infected with Pasteurella multocida. The results showed that the optimal reaction temperature was 39 ℃, the optimal primer was KMT1-Fe1, and the sensitivity was 100 copies/μL. The detection limit was 10 copies/μL. There was no cross reaction with Escherichia coli, Staphylococcus aureus, Salmonella Paratyphi, Vibrio parahaemolyticus, Pseudomonas aeruginosa, Klebsiella acidogenesis, Brucella S2 and Acinetobacter baumannii. The positive rate of 13 tissue samples was 76.9%, and 92.3% of them were consistent with Real-time PCR. In conclusion, the Real-time RPA of Pasteurella multocida from goat had the characteristics of strong specificity, high sensitivity, good reliability, fast and convenient, which was suitable for clinical molecular diagnosis of Pasteurella multocida.

Key words: Pasteurella multocida; KMT1 gene; RPA detection; fluorescent probe; molecular diagnosis