Dual Real-time PCR Detection of methicillin-resistant Staphylococcus aureus_3012

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Dual Real-time PCR Detection of methicillin-resistant Staphylococcus aureus_3012

Dual Real-time PCR Detection of methicillin-resistant

    Staphylococcus aureus

     Abstract Objective To establish the two heavy real-time

    polymerase chain reaction (duplex real time PCR) Rapid

    detection of methicillin-resistant Staphylococcus aureus

    (MRSA). Methods The two re-SYBR Green real-time PCR for rapid

    detection of MRSA determining gene mecA and Staphylococcus aureus species-specific gene nuc, identified by melting curve analysis product. The results of the melting curve of all MRSA

    strains showed a mecA, nuc gene-specific peaks, methicillin-

    sensitive Staphylococcus aureus only nuc-feng, methicillin-

    resistant strains of the table Portuguese only mecA-feng,

    methicillin-sensitive tables Portuguese bacteria and other bacteria strains non-specific peaks; when the MRSA bacteria can be detected when the concentration of 102cfu/ml. In the 131 S. aureus in 30.53% (40/131) resistance; two heavy real-

    time PCR amplification of mecA gene 29.01% (38/131) positive, nuc gene 100% positive. Single primer and two heavy real-time

    PCR amplification of both the positive coincidence rate of 100%. Conclusion mecA, nuc genes of two re-real-time PCR can

    accurately and quickly identify methicillin-resistant

    Staphylococcus aureus and coagulase-negative staphylococci.

     Key words real-time polymerase chain reaction of methicillin-

    resistant Staphylococcus aureus mecA gene nuc gene

     Rapid detection of methicillin resistant

    Staphylococcus aureus

     ABSTRACT Objective To establish a duplex real time

    polymerase chain reaction (PCR) assay for rapid detection of methicillin resistant Staphylococcus aureus (MRSA). Method A duplex SYBR Green real time PCR assay targeting the mecA

gene and a Staphylococcus aureus specific marker nuc gene

    and identifying PCR products through melting curve analysis was used to rapidly identify MRSA. Results All MRSA strains tested in the study presented mecA and nuc gene specific peaks in the melting curve analysis; methicillin susceptible

    Staphylococcus aureus (MSSA) strains only had a nuc peak, methicillin resistant Staphylococcus epidermidis (MRSE) strains only had a mecA peak, and methicillin susceptible

    Staphylococcus epidermidis (MSSE) strains and strains of species other than staphylococci had no peak. MRSA strains could be detected, by use of this duplex real time PCR in

    an 102 cfu / ml inoculum. Positive rate of drug sensitivity

    test was 30.53% (40/131) among 131 S.aureus isolates tested, of which 29.01% (38/131) were positive for mecA gene and all were positive for nuc gene in the duplex real time PCR. The

    agreement between simplex and duplex real time PCR was 100%

    for positive results. Conclusion This study shows that the duplex real time PCR assay with the primers for to mecA and nuc genes is a valuable tool for rapid and precise identification of MRSA and methicillin resistant coagulase

     negative staphylococci (MRCNS).

     KEY WORDS Real time polymerase chain reaction (RT

    PCR); Methicillin resistant Staphylococcus aureus (MRSA); mecA gene; nuc gene Received Date: 2006 06 15 Revised:

    2006 10 30

     Staphylococcus aureus is caused by hospitals and community-acquired infections important human pathogen, in integration of staphylococcal cassette chromosome mec element (staphylococcal cassette chromosome mec element, SCCmec) and