Detection of Methicillin-ResistantStaphylococcus aureusand Simultaneous Confirmation by Automated Nucleic Acid Extraction and Real-Time PCR

Abstract

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.

Sensitivity and Specificity of a Rapid rRNA Gene Probe Assay for Simultaneous Identification of Staphylococcus aureus and Detection of mecA

“…Recently, molecular assays for methicillin resistance and Staphylococcus identification using mecA and Staphylococcusspecific genes have yielded fast and reliable results (4,6,9,12,13,16,20). Molecular assays have an advantage because they do not rely on time-consuming incubations or phenotypic expression (7).…”

“…Rapid identification of Staphylococcus, more specifically S. aureus, along with the methicillin resistance status has been the focus of many recent studies (4,6,9,12,13,20). Many direct and amplified formats have problems with false negatives due to lack of an internal control to verify that sufficient target was present and accessible.…”

Rapid Identification of Bacteria from Positive Blood Cultures by Terminal Restriction Fragment Length Polymorphism Profile Analysis of the 16S rRNA Gene

“…Previous reports have confirmed a high correlation between production of coagulase and an extracellular thermostable nuclease, encoded by nuc in S. aureus (3,17,18). In addition, the presence of mecA strongly correlates with methicillin resistance (13,(16)(17)(18). Therefore, with the current assay design, it is possible to rapidly and accurately identify staphylococci, predict the coagulase phenotype, and predict methicillin resistance on the basis of TRF sizes from HhaI digests of products from two PCR samples.…”

Detection of Methicillin-Resistant Staphylococcus aureus by a Duplex Droplet Digital PCR Assay

“…The superior sensitivity and specificity of PCR has made it the method of choice for the rapid detection of MRSA in a clinical setting (17,18). A duplex PCR assay was designed that combined a set of primers for chromosomal mecA and an unlinked S. aureusspecific gene (SA0140) for the detection of MRSA.…”