Staphylococcus aureus(S.aureus),Pseudomonas aeruginosa(P.aeruginosa),Bordetella bronchiseptica(B.bronchiseptica),Mycoplasma pneumoniae(M.pneumoniae),Pasteurella multocida(P.multocida)are commune pathogens isolated frequently in laboratory animals.Various diagnostic methods have been developed to identify these pathogens.In this report,we developed and evaluated a multiplex PCR for detecting these five pathogens in laboratory animals simultaneously,which is significant in differential diagnosis and screening.The primer concentrations and PCR conditions were optimized.The sensitivity of the multiplex PCR for simultaneous amplification is 10 pg of DNA per target.The specificity of the PCR was demonstrated by detecting pathogen-spiked clinical samples.A total of 182 clinical specimens were tested by the multiplex PCR,and the results were consistent with the results obtained using conventional bacterial isolation methods.In conclusion,the multiplex PCR developed in this report is a specific and sensitive method that can be used for differential diagnosis of five bacterial pathogen infections and screening in laboratory animals.