Abstract: Objective To develop a mutiplex real-time PCR for the detection of specific structural genes and virulence genes of Bacillus anthracis,Yersinia pestis,and Legionella pneumophila.Methods Six pairs of specific primers and six fluorogen-labled probes were designed and synthesized according to capA and PA genes of Bacillus anthracis,pla and caf1 genes of Yersinia pestis,and pilE and Mip genes of Legionella pneumophila.The reaction parameters such as the concentration of primers,probes and the reaction buffer were optimized to develop two sets of multiplex real-time PCR assay for rapid detection of Bacillus anthracis,Yersinia pestis,and Legionella pneumophila simultaneously.By using the same method,the duplex real-time PCR for the detection of Bacillus anthracis,Yersinia pestis,and Legionella pneumophila were also estimated.Results The detectable concentration for the multiplex real-time PCR and duplex real-time PCR was 100 template copy per reaction and 20 template copy per reaction,respectively,and the detection had good specificity,stability,and reproducibility.Conclusion The multiplex real-time fluorescence quantitative PCR assay developed in the study has good specificity and sensitivity and could to be applied for the detection of Bacillus anthracis,Yersinia pestis,and Legionella pneumophila.