title: Multiplex real-time PCR assay for rapid detection of methicillin-resistant staphylococci directly from positive blood cultures.
abstract: Methicillin-resistant Staphylococcus aureus (MRSA) is the most prevalent cause of bloodstream infections (BSIs) and is recognized as a major nosocomial pathogen. This study aimed to evaluate a newly designed multiplex real-time PCR assay capable of the simultaneous detection of mecA, S. aureus, and coagulase-negative staphylococci (CoNS) in blood culture specimens. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays (M&D, Republic of Korea) use the TaqMan probes 16S rRNA for Staphylococcus spp., the nuc gene for S. aureus, and the mecA gene for methicillin resistance. The detection limit of the multiplex real-time PCR assay was 10(3) CFU/ml per PCR for each gene target. The multiplex real-time PCR assay was evaluated using 118 clinical isolates from various specimen types and a total of 350 positive blood cultures from a continuous monitoring blood culture system. The results obtained with the multiplex real-time PCR assay for the three targets were in agreement with those of conventional identification and susceptibility testing methods except for one organism. Of 350 positive bottle cultures, the sensitivities of the multiplex real-time PCR kit were 100% (166/166 cultures), 97.2% (35/36 cultures), and 99.2% (117/118 cultures) for the 16S rRNA, nuc, and mecA genes, respectively, and the specificities for all three targets were 100%. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays are very useful for the rapid accurate diagnosis of staphylococcal BSIs. In addition, the Real-MRSA and Real-MRCoNS multiplex real-time PCR assays could have an important impact on the choice of appropriate antimicrobial therapy, based on detection of the mecA gene.
authors.affiliation: M&D, Inc., Wonju, Republic of Korea.
authors.forename: Hye-Young
authors.initials: HY
authors.lastname: Wang
authors.affiliation: Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju, Republic of Korea Institute for Life Science and Biotechnology, Yonsei University, Seoul, Republic of Korea.
authors.forename: Sunghyun
authors.initials: S
authors.lastname: Kim
authors.affiliation: Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju, Republic of Korea.
authors.forename: Jungho
authors.initials: J
authors.lastname: Kim
authors.affiliation: Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju, Republic of Korea Department of Laboratory Medicine, Wonju College of Medicine, Yonsei University, Wonju, Republic of Korea.
authors.forename: Soon-Deok
authors.initials: SD
authors.lastname: Park
authors.affiliation: Department of Laboratory Medicine, Wonju College of Medicine, Yonsei University, Wonju, Republic of Korea u931018@yonsei.ac.kr hyelee@yonsei.ac.kr.
authors.forename: Young
authors.initials: Y
authors.lastname: Uh
authors.affiliation: Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju, Republic of Korea u931018@yonsei.ac.kr hyelee@yonsei.ac.kr.
authors.forename: Hyeyoung
authors.initials: H
authors.lastname: Lee
mesh_terms: D016470:Bacteremia / Q000175:diagnosis* / Q000382:microbiology; D001426:Bacterial Proteins / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D006801:Humans; D055624:Methicillin-Resistant Staphylococcus aureus / Q000235:genetics / Q000302:isolation & purification*; D008836:Micrococcal Nuclease / Q000235:genetics; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D046915:Penicillin-Binding Proteins; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013203:Staphylococcal Infections / Q000175:diagnosis* / Q000382:microbiology
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D046915:Penicillin-Binding Proteins; D012336:RNA, Ribosomal, 16S; C487789:mecA protein, Staphylococcus aureus; C074002:nuc protein, staphylococcus; D008836:Micrococcal Nuclease