Establishment of a Multiplex Molecular Test Method for Three Types of Conditional Pathogenic Bacteria in Laboratory Animals
Objective 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.
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