Double triplex real-time PCR assay for simultaneous detection of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, and Staphylococcus haemolyticus and determination of their methicillin resistance directly from positive blood cult

We developed and validated here a double triplex real-time PCR assay to simultaneously detect and identify Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus haemolyticus and their methicillin resistance in a single reaction directly from Gram-positive cocci-in-clusters (GPCs)-positive blood culture bottles. From August 15, 2009 through February 15, 2010, 238 GPC-positive samples were collected and identified by conventional methods as 11 methicillin-resistant S. aureus (MRSA), 28 methicillin-susceptible S. aureus (MSSA), 176 MR coagulase-negative staphylococci (MRCoNS), 21 MSCoNS and two Enterococcus faecalis. The double triplex real-time PCR assay was targeted and detected tuf, nuc and mecA genes in the first tube and atlE, gap and mvaA genes in the second tube which could be run simultaneously. The detection limit of the assay was found at 10(3) CFU/ml for the atleE gene, 10(4) CFU/ml for the mva gene and 10(5) CFU/ml for gap, nuc, mecA and tuf genes based on seeding experiments. All Staphylococcus species except two S. epidermidis were correctly identified by the assay. The double triplex real-time PCR assay quickly and accurately detects S. aureus, S. epidermidis, S. hominis and S. haemolyticus and their methicillin resistance in a single reaction directly from positive blood culture bottles within 83 min.

Kilic A ，Basustaoglu AC 《-》

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 A ，Muldrew KL ，Tang YW ，Basustaoglu AC ... -  《-》

Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus haemolyticus: methicillin-resistant isolates are detected directly in blood cultures by multiplex PCR.

Pereira EM ，Schuenck RP ，Malvar KL ，Iorio NL ，Matos PD ，Olendzki AN ，Oelemann WM ，dos Santos KR ... -  《-》

Multiplex PCR assay to identify methicillin-resistant Staphylococcus haemolyticus.

Schuenck RP ，Pereira EM ，Iorio NL ，Dos Santos KR ... -  《fems immunology and medical microbiology》

StaphPlex system for rapid and simultaneous identification of antibiotic resistance determinants and Panton-Valentine leukocidin detection of staphylococci from positive blood cultures.

Tang YW ，Kilic A ，Yang Q ，McAllister SK ，Li H ，Miller RS ，McCormac M ，Tracy KD ，Stratton CW ，Han J ，Limbago B ... -  《-》