Rapid detection of methicillin-resistant Staphylococci by multiplex PCR

Kearns, Angela; Seiders, P.R.; Wheeler, Jenna; Freeman, R.; Steward, M. W.

doi:10.1053/jhin.1999.0631

Cited by 71 publications

(47 citation statements)

References 16 publications

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“…Numerous molecular approaches that reduce the time for identification of MRSA have been described (3,12,13,26,30,35,42,46,48,49). However, the molecular tests developed to date for the detection of MRSA are based on the detection of an S. aureusspecific gene and/or mecA.…”

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confidence: 99%

New Real-Time PCR Assay for Rapid Detection of Methicillin- ResistantStaphylococcus aureusDirectly from Specimens Containing a Mixture of Staphylococci

et al. 2004

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Molecular methods for the rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) are generally based on the detection of an S. aureus-specific gene target and the mecA gene. However, such methods cannot be applied for the direct detection of MRSA from nonsterile specimens such as nasal samples without the previous isolation, capture, or enrichment of MRSA because these samples often contain both coagulasenegative staphylococci (CoNS) and S. aureus, either of which can carry mecA. In this study, we describe a real-time multiplex PCR assay which allows the detection of MRSA directly from clinical specimens containing a mixture of staphylococci in <1 h. Five primers specific to the different staphylococcal cassette chromosome mec (SCCmec) right extremity sequences, including three new sequences, were used in combination with a primer and three molecular beacon probes specific to the S. aureus chromosomal orfX gene sequences located to the right of the SCCmec integration site. Of the 1,657 MRSA isolates tested, 1,636 (98.7%) were detected with the PCR assay, whereas 26 of 569 (4.6%) methicillin-susceptible S. aureus (MSSA) strains were misidentified as MRSA. None of the 62 nonstaphylococcal bacterial species or the 212 methicillin-resistant or 74 methicillinsusceptible CoNS strains (MRCoNS and MSCoNS, respectively) were detected by the assay. The amplification of MRSA was not inhibited in the presence of high copy numbers of MSSA, MRCoNS, or MSCoNS. The analytical sensitivity of the PCR assay, as evaluated with MRSA-negative nasal specimens containing a mixture of MSSA, MRCoNS, and MSCoNS spiked with MRSA, was ϳ25 CFU per nasal sample. This real-time PCR assay represents a rapid and powerful method which can be used for the detection of MRSA directly from specimens containing a mixture of staphylococci.

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confidence: 99%

New Real-Time PCR Assay for Rapid Detection of Methicillin- ResistantStaphylococcus aureusDirectly from Specimens Containing a Mixture of Staphylococci

et al. 2004

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“…In the case of severe infections, it is clinically useful to provide rapid identification and antimicrobial susceptibility test results (5). Many multiplex PCR techniques have been described for the characterization of staphylococci (4,13,14,25,27). Most of these are performed with genomic DNA extracts from bacterial colonies or broth culture lysates.…”

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confidence: 99%

Evaluation of a Triplex PCR Assay To DiscriminateStaphylococcus aureusfrom Coagulase-Negative Staphylococci and Determine Methicillin Resistance from Blood Cultures

et al. 2002

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A triplex PCR targeting the 16S rRNA, mecA, and nuc genes was developed for identification of staphylococci and detection of methicillin resistance. After validation of the assay with a collection of strains of staphylococci and enterococci (n ‫؍‬ 169), the assay was evaluated with cultures of blood with gram-positive cocci from 40 patients. Accurate results were obtained for 59 (98%) of 61 cultures within 6 h of growth detection.

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“…A variation using the S. aureus-specific coa (coagulase) gene was described recently (145). Complete agreement was observed between the results of the coa-specific PCR and conventional slide coagulase testing with S. aureus and CNS isolates.…”

Section: Methicillin-resistant Staphylococcimentioning

confidence: 78%