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.
Kilic
Abdullah
A
Department of Microbiology and Clinical Microbiology, Gulhane Military Medical Academy and School of Medicine 06018, Ankara, Turkey. abkilic@gata.edu.tr
Muldrew
Kenneth L
KL
Tang
Yi-Wei
YW
Basustaoglu
A Celal
AC
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