ObjectiveTo establish a multiplex PCR procedure for simultaneous detection of H. hepaticus, H. bilis, and H. rodentium, and to preliminarily verify its application value.Methods Three pairs of specific primers for the multiplex PCR procedure were designed according to published H. hepaticus, H. bilis, and H. rodentium 16S rRNA gene sequences, and the reaction conditions of the multiplex PCR were optimized. ResultsThe results showed that all the three pairs of primers could be used to specifically amplify their target bands of 417 bp, 364 bp and 324 bp. The optimal annealing temperature was 52℃, the Mg²⁺ concentration was 2.0 mM, the dNTP concentration was 200 μΜ, and the concentration of primers was 0.625 μM. The sensitivity of the multiplex PCR for H. hepaticus, H. bilis, H. rodentium was 10 fg. CouclusionsA sensitive, specific, efficient multiplex PCR procedure has been established in this study, providing a good foundation for the detection of H. hepaticus, H. bilis, and H. rodentium.