Triplex real-time polymerase chain reaction assay for simultaneous detection of Staphylococcus aureus and coagulase-negative staphylococci and determination of methicillin resistance directly from positive blood culture bottles

• Published in: Diagnostic microbiology and infectious disease Vol. 66; no. 4; pp. 349 - 355
• Main Authors: Kilic, Abdullah, Muldrew, Kenneth L, Tang, Yi-Wei, Basustaoglu, A. Celal
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-04-2010
• Subjects: Bacterial Proteins - genetics; Bacteriological Techniques - methods; Blood - microbiology; Humans; Infectious Disease; Internal Medicine; Methicillin Resistance; Micrococcal Nuclease - genetics; MRCoNS; MRSA; MSCoNS; MSSA; Peptide Elongation Factor Tu - genetics; Polymerase Chain Reaction - methods; Sensitivity and Specificity; Staphylococcal Infections - diagnosis; Staphylococcal Infections - microbiology; Staphylococcus - classification; Staphylococcus - drug effects; Staphylococcus - genetics; Staphylococcus - isolation & purification; Staphylococcus aureus; Triplex PCR; MSSA; Triplex PCR; MSCoNS; MRCoNS; MRSA
• ISSN: 0732-8893; 1879-0070
• DOI: 10.1016/j.diagmicrobio.2009.11.010

Abstract We describe here a 1-step, triplex real-time polymerase chain reaction (PCR) assay for the detection and identification of staphylococci directly from signal-positive blood culture bottles containing Gram-positive cocci in clusters (GPCC). The triplex assay targeted and detected tuf , nuc , and mecA genes in a single tube and had a detection limit of 105 CFU/mL for each gene target. A total of 341 GPCC-positive blood culture bottles were collected between November 12, 2008, and August 11, 2009. Among them, 230 methicillin-resistant coagulase-negative staphylococci (CoNS), 54 methicillin-susceptible CoNS, 22 methicillin-resistant Staphylococcus aureus , 22 methicillin-susceptible S. aureus , and 13 nonstaphylococci species were identified by conventional methods. The results obtained by triplex assay were in agreement with those of conventional methods for tuf (99.7%), nuc (100.0%), and mecA (99.1%), respectively. The triplex assay was found to have sensitivities of 99.7%, 100%, and 99.2% and specificities of 100%, 100%, and 98.7%, respectively, for the tuf , nuc , and mecA gene targets. The triplex real-time PCR assay accurately detects and identifies staphylococci directly from positive blood cultures without nucleic acid extraction prior to amplification.