Detection of methicillin-resistant Staphylococcus aureus and simultaneous confirmation by automated nucleic acid extraction and real-time PCR.
A molecular assay for the simultaneous detection of a Staphylococcus aureus-specific gene and the mecA gene, responsible for the resistance to methicillin in staphylococci, was evaluated. The assay included an automated DNA extraction protocol conducted with a MagNA Pure instrument and real-time PCR conducted with a LightCycler instrument. The performance and robustness of the assay were evaluated for a suspension of methicillin-resistant S. aureus (MRSA) strain with a turbidity equivalent to a McFarland standard of 0.5, which was found to be the ideal working concentration. The specificity of the new molecular assay was tested with a panel of 30 gram-negative and gram-positive bacterial strains other than MRSA. No cross-reactivity was observed. In a clinical study, 109 isolates of MRSA were investigated. All clinical MRSA isolates gave positive results for the S. aureus-specific genomic target, and all but one were positive for the mecA gene. In conclusion, the new molecular assay was found to be quick, robust, and laborsaving, and it proved to be suitable for a routine molecular diagnostic laboratory.
Grisold
Andrea J
AJ
Institute of Hygiene, Karl Franzens University Graz, A-8010 Graz. Roche Diagnostics GmbH, A-1211 Vienna, Austria.
Leitner
Eva
E
Mühlbauer
Gerhard
G
Marth
Egon
E
Kessler
Harald H
HH
D001426:Bacterial Proteins; D001431:Bacteriological Techniques; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D005798:Genes, Bacterial; D006602:Hexosyltransferases; D006801:Humans; D016106:Methicillin Resistance / Q000235:genetics; D002267:Muramoylpentapeptide Carboxypeptidase / Q000235:genetics; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D016133:Polymerase Chain Reaction / Q000379:methods*; D013203:Staphylococcal Infections / Q000175:diagnosis / Q000188:drug therapy / Q000382:microbiology; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification
D001426:Bacterial Proteins; D002352:Carrier Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase