动物造模,实验动物,PCR z-bio 2022-03-12 635

　　Objective: To establish a multiplex PCR method for the rapid detection of four experimental animal pathogens, Pasteurella multocida, Bordetella bronchiscus, Mycoplasma and Klebsiella pneumoniae.

　　Methods: Specific primers were designed according to GenBank genes; after the optimization of multiplex PCR, the detection of specificity and sensitivity, a multiplex PCR system was established. The PCR system was used to detect the tracheal secretions of artificially infected samples and experimental animals, and compared with traditional methods.

　　RESULTS: Multiplex PCR amplified the target bands of Pasteurella multocida (356 bp), Bordetella bronchiscus (237 bp), Mycoplasma (266 bp) and Klebsiella pneumoniae (142 bp). The sensitivity of Klebsiella pneumoniae was 10 pg, and the sensitivity of Pasteurella multocida, Bordetella bronchus, and Mycoplasma was 1 pg. The specific detection did not detect the target band from other pathogens. Using the established multiplex PCR system to detect different combinations of artificially infected samples, 15 were positive for Pasteurella multocida and 9 were positive for Mycoplasma pulmonary in the trachea of 45 experimental animals, but no positive samples were detected by traditional culture methods and serological methods.

　　Conclusion: The multiplex PCR method established in this paper is simple, rapid, specific and sensitive, and can achieve rapid detection of four experimental animal pathogens: Pasteurella multocida, Bordetella bronchus, Mycoplasma and Klebsiella pneumoniae.