The mecA gene, the structural determinant that encodes PBP2a, is therefore considered as a useful molecular marker of putative methicillin resistance in S. aureus and CoNS [9, 10]. Clinical laboratory tests

for methicillin resistance are highly dependent on growing conditions such as temperature, pH and salt concentration [11]. Thus, these factors emphasize the need to develop a rapid, accurate and sensitive method for detection of methicillin-resistant staphylococci, which does not depend on growth conditions. Nucleic-acid-based tests using PCR are increasingly being used in laboratories to replace time-consuming, labor intensive and less sensitive conventional diagnostic methods, such as biochemical identification and Kirby-Bauer antimicrobial susceptibility tests. Various PCR methods have been developed to identify: (i) Staphylococcus genus [12]; (ii) methicillin-resistance selleck screening library [13]; and (iii) Panton-Valentine leukocidin (PVL)-producing Staphylococcus genus [14]. These methods do not detect all of the above-mentioned targets simultaneously. Hence, the present study focused on the design of a pentaplex PCR for methicillin-resistant staphylococci with an internal control for the detection of Staphylococcus genus (16S rRNA gene), methicillin-resistant staphylococci (mecA gene), community-acquired

MRSA (lukS gene), and discrimination between S. aureus and CoNS BCKDHB (femA gene). Results In the present study, the pentaplex PCR was optimized successfully to identify the Staphylococcus genus (16S check details rRNA), S. aureus species (femA), methicillin resistance (mecA) and PVL toxin (lukS) genes simultaneously. Stepwise optimization of primer concentration, annealing temperature, MgCl2, dNTP and Taq polymerase was carried out. The pentaplex PCR gave the best results

when 3.13 mM MgCl2, 200 μM dNTP, 0.75 U Taq polymerase and 60°C annealing temperature were used. The analytical sensitivity of the pentaplex PCR at the DNA level was found to be 10 ng DNA (data not shown), whereas, at the bacterial level, it was found to be 104 CFU/mL (data not shown). The analytical specificity of the pentaplex PCR assay at the genus level was determined using 10 staphylococcal reference strains and found to be positive for the Staphylococcus genus specific 16S rRNA gene. A representative gel picture of methicillin resistance with reference strains is shown in Figure 1, while the other 10 Gram-positive non-staphylococcal and 13 Gram-negative strains were negative. All the reference strains of S. aureus were positive for femA gene by pentaplex PCR, while other CoNS species were negative (Table 1). Hence, all methicillin-resistant reference strains were positive for mecA gene by pentaplex PCR. However, the methicillin-sensitive reference strains were negative for mecA gene by pentaplex PCR (Table 1).