关键词: 多杀性巴氏杆菌, 金黄色葡萄球菌, 肺炎克雷伯菌, 多重 PCR

Abstract: Objective This study aimed to establish a multiplex PCR method for detecting three pathogens in rabbits: Pasteurella multocida ( Pm ) , Staphylococcus aureus ( Sa ) , and Klebsiella pneumoniae (Kp) . Method In this study, three pairs of specific primers were designed, targeting the conserved regions of the kmt1 gene in Pm, the nuc gene in Sa, and the kh1 gene in Kp. The optimal annealing temperature ( Tm ) was determined using gradient PCR. The primer concentrations were optimized using the controlled single-variable method. Recombinant plasmid standards ( pMD-Pm, pMDSa, and pMD-Kp) were constructed, and the minimum detection limit was established. The specificity of the multiplex PCR was confirmed using nucleic acid samples of nine pathogens, including rabbit bronchial septicemic Pasteurella and gas-producing clostridia. The repeatability was validated through inter-batch and intra-batch experiments. Clinical samples suspected of infection were tested using the established method and compared with previously reported detection methods to evaluate its clinical application. Result The optimal annealing temperature was determined to be 54. 5 ℃ . The optimal primer concentrations for amplifying kmt1 and nuc genes were 1 μL ( 10 μmol / L) , while for the kh1 gene, it was 0. 5 μL (10 μmol / L) . The lowest detection limits were 20. 6 copies/ μL for pMD-Pm, 18. 2 copies/ μL for pMD-Sa, and 23. 2 copies/ μL for pMD-Kp, indicating high sensitivity. The multiplex PCR method showed specific amplification bands only for Pm, Sa, and Kp, with no amplification bands observed for other pathogens, demonstrating strong specificity. The results of inter-batch and intra-batch tests were consistent, indicating good repeatability. Clinical sample testing revealed individual positivity rates of 62. 92% for Pm, 25. 84% for Sa, and 49. 43% for Kp, which were consistent with the results obtained using previously reported detection methods. Conclusion This study successfully established a multiplex PCR method capable of simultaneously and rapidly detecting Pm, Sa, and Kp in rabbits.

Key words: Pasteurella multocida, Staphylococcus aureus, Klebsiella pneumoniae, duplex PCR