Abstract: To establish a multiplex PCR assay for simultaneous detection of Klebsiella pneumoniae, Acinetobacter johnsonii, Pasteurella multocida of beef cattle, specific primers were designed based on Khe gene of Klebsiella pneumoniae, Ptk gene of Acinetobacter johnsonii, and Abhd gene of Pasteurella multocida. The reaction conditions and system of the multiplex PCR detection method were established by specificity test, sensitivity test, primer concentration test and annealing temperature test. The results showed that the method can simultaneously amplify Klebsiella pneumoniae, Acinetobacter johnsonii and Pasteurella multocida, and the similarity between bacterial sequences of three kinds of target bacteria amplification and sequencing on GenBank were more than 99%. Meanwhile, the amplification results of the other 10 kinds of beef respiratory pathogens were all negative. The detection limits of the above three pathogen genomic DNA were 69.8×10^-5, 208.9×10^-4, 70.8×10^-3 ng·μL^-1, and the optimal primer concentration ratio was 1:1:1. The optimum annealing temperature was 58 ℃. The method had a strong specificity and high sensitivity, and provided a convenient, rapid and accurate tool for rapid detection and identification of the above three pathogens and diagnosis of clinical bovine respiratory diseases.

Key words: beef cattle respiratory tract, Klebsiella pneumoniae, Acinetobacter johnsonii, Pasteurella multocida, multiplex PCR