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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 by Jürgen Weidner, Uwe Cassens, Wolfgang Göhde, Jörg Wüllenweber, Burkhard Greve

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°C for the mecA gene, 83.15°C for the SCCmec/orfX fragment and 88.25°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.

DOI: 10.7754/Clin.Lab.2012.120706