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.
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 10(5) 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.
Department of Microbiology and Clinical Microbiology, Gulhane Military Medical Academy and School of Medicine 06018, Ankara, Turkey. abkilic@gata.edu.tr
Abdullah
A
Kilic
Kenneth L
KL
Muldrew
Yi-Wei
YW
Tang
A Celal
AC
Basustaoglu
D001426:Bacterial Proteins / Q000235:genetics; D001431:Bacteriological Techniques / Q000379:methods*; D001769:Blood / Q000382:microbiology*; D006801:Humans; D016106:Methicillin Resistance; D008836:Micrococcal Nuclease / Q000235:genetics; D010444:Peptide Elongation Factor Tu / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013203:Staphylococcal Infections / Q000175:diagnosis* / Q000382:microbiology; D013210:Staphylococcus / Q000145:classification / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification*
D001426:Bacterial Proteins; C074002:nuc protein, staphylococcus; D008836:Micrococcal Nuclease; D010444:Peptide Elongation Factor Tu