title: A new triplex real time PCR which distinguishes between MRSA, MSSA, and mecA coagulase negative strains by means of melting point analysis using SYTO 9.
abstract: BACKGROUND
  A new screening method was developed for the detection and identification of methicillin resistant staphylococcus aureus (MRSA) from sterile sites or mixed flora samples (inguinal or nose swabs).
  
  
  METHODS
  After rapid treatment of samples, the method consists of two steps, template DNA preparation by a simple and rapid boiling procedure and a multiplex real time PCR. The triplex PCR system simultaneously detects the following targets, (i) the integration site for the open reading frame X (orfX) of the staphylococcal cassette chromosome mec type I - V (SCCmec), (ii) the mecA gene which codes for the penicillin-binding protein PBP-2a, and (iii) an internal control (IC) which can be amplified with the SCCmec primer system. A new buffer system, which contains the fluorescent dye SYTO 9, allows a reproducible real time PCR for the discrimination of the above mentioned PCR products by means of a high resolution melting point analysis (HRM).
  
  
  RESULTS
  This new PCR system distinguishes between MRSA, MSSA, and mecA positive but coagulase-negative staphylococci (CoNS) strains. An internal control confirms the integrity of the PCR run. The HRM shows three melting points specific for each amplification product. 78.75 degrees C for the mecA gene, 83.15 degrees C for the SCCmec/orfX fragment and 88.25 degrees C for the internal control.
  
  
  CONCLUSIONS
  This new multilocus MRSA PCR system is a fast and inexpensive alternative to commercially available test systems and costs only five to six euros.
authors.affiliation: Department of Radiotherapy, University Hospital Münster, Albert-Schweitzer Campus 1, Münster, Germany. j.weidner@partec.com
authors.forename: Jürgen
authors.initials: J
authors.lastname: Weidner
authors.forename: Uwe
authors.initials: U
authors.lastname: Cassens
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Göhde
authors.forename: Jörg
authors.initials: J
authors.lastname: Wüllenweber
authors.forename: Burkhard
authors.initials: B
authors.lastname: Greve
mesh_terms: D003030:Coagulase / Q000378:metabolism*; D057230:Limit of Detection; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013211:Staphylococcus aureus / Q000145:classification / Q000201:enzymology / Q000235:genetics*
chemical_list: D003030:Coagulase