Abstract: A multiplex qPCR( Fluorescence quantitative PCR, qPCR) test method for three
conditional pathogens, Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa, was
established. Method The nuc gene of Staphylococcus aureus, the phoE gene of Klebsiella pneumoniae,
and the gyrB gene of Pseudomonas aeruginosa were selected to design specific primers and probes, and a
multiplex qPCR method was established. Its performance indicators such as specificity, sensitivity,
standard curve, and repeatability were tested. This method was used to test 30 clinical stool samples and
6 positive references. The test result were compared with the result of the culture method, and the
compliance rate was calculated. Result The multiplex qPCR method can stably test the genomic DNA of
standard strains of Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa; there is
no nonspecific amplification of pathogenic microorganisms such as Salmonella typhimurium, Pasteurella
pneumotropica, Corynebacterium kutscheri, and Bordetella bronchiseptica; the sensitivity of the genomic
DNA of standard strains of Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa is
1. 0×10^{- 5},1. 0×10^{- 4},and 1. 0×10^{- 5}ng / μL, respectively; the inter-batch and intra-batch differences are less than 2%, and the repeatability is good; the test result of clinical samples and positive references
show that the test result of the multiplex qPCR method are consistent with those of the isolation and
culture method. Conclusion This study successfully established a multiplex qPCR method for
simultaneous and rapid detection of Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas
aeruginosa.

Key words: multiplex real-time PCR method, culture method, conditional pathogenic bacteria

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