Findings To facilitate automation, a novel DNA extraction method for MRSA was adopted. MRSA detection kits were added to our PCR 2152-44-5 to detect all currently known variant SCC-mec types of MRSA. Background The dissemination of Methicillin Resistant Staphylococcus aureus (MRSA) in hospitals is a growing problem worldwide. In The Netherlands, a search and destroy policy is implemented . Patients colonized with MRSA are kept in isolation until they are culture negative. A MRSA negative report can faster be obtained by PCR. Therefore, a molecular approach for negative screening of MRSA was exploited. Molecular detection of the mecA gene, which confers resistance to all -lactams, has often been used in combination with other S. aureus specific genes in a multiplex PCR. Genes that are specific for S. aureus comprise for example of the sequence published by Martineau et al. , the nuclease gene (nuc) [3,4], or the coagulase gene (coa) . When clinical samples contain 2152-44-5 a mixture of coagulase negative staphylococci (CNS), methicillin sensitive S. aureus (MSSA), and MRSA, a positive mecA PCR can be generated by CNS while both MSSA and MRSA generate a positive PCR for the coa or the nuc gene. Only culturing could confirm MRSA. Another approach for detection of MRSA was presented by a multiplex PCR described by Huletsky et al. . This PCR specifically targets the junction between a conserved open reading frame orfX in S. aureus, and the staphylococcal cassette chromosome containing the mecA gene (SCCmec). For MRSA, 8 different types of SCCmec elements have been classified . The SCC is known to be a mobile heterogeneous genetic element that integrates site Rabbit polyclonal to POLR3B specifically into orfX. SCCmec is an SCC containing the mecA gene. SCC can also be present in CNS, not containing mecA but integrated into the analogous chromosomal location. MSSA can contain non-mecA-SCC or SCCmec elements which have lost the region containing mecA. Several commercially available molecular screening tests are based on PCR amplification of the chromosome-SCCmec junction. In this study, a novel DNA extraction method for MRSA was adopted that virtually prevents PCR inhibition. The detection process was fully automated 2152-44-5 for high through-put of clinical materials. An extra 17 forward primers were added to PCR to detect several newly identified MRSA strains in this study carrying SCCmec variants and found to be present in The Netherlands, and possibly elsewhere. An adaptable PCR format is needed for reliable detection of all MRSA. Findings Implementation and evaluation of orfX/SCCmec PCR The PCR as described by Huletsky et al. , was slightly adapted (Table ?(Table11). Table 1 Primer and probe sequences used in OrfX-SCC PCR To allow PCR detection of more MRSA types, a literature search was conducted. Forward primer F7 was derived from the sequence of S. aureus strain JCSC 3624 (WIS), accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”AB121219″,”term_id”:”49257031″,”term_text”:”AB121219″AB121219 , and was included in the PCR. Primer F10 was designed based on the sequence of S. aureus “type”:”entrez-nucleotide”,”attrs”:”text”:”U10927″,”term_id”:”16579831″,”term_text”:”U10927″U10927  (Table ?(Table11). With the expanded orfX/SCCmec PCR a total of 1906 samples was investigated with high through-put screening; 303 were PCR positive, no inhibition of PCR was found. To verify whether a positive signal was due to viable or dead MRSA, all were cultured; 141 were culture positive, and 22% of 141 were found to be MSSA. The latter may have lost mecA.