[Colony PCR for rapid detection of antibiotic resistance genes in MRSA and enterococci]

We examined colony PCR that does not use the prepared template DNA but intact cells as the template DNA source for the rapid, simple and reproducible detection of antibiotic resistance genes in strains of methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus. Two factors turned out to be important; i.e. the transfer size of bacterial cells and the use of DNA Polymerase with high performance. In practice, the tip of a toothpick was lightly touched with a colony on agar plates followed by the PCR mixture (20 microliters) containing KOD-plus-DNA Polymerase and multiplex primers. The cell transfer size ranged from 10(3) to 10(4) cfu and 30 cycles of PCR (95 degrees C, 30 sec.-->50 degrees C, 30 sec.-->72 degrees C, 60 sec.) following the first heating (95 degrees C, 3 min.) resulted in good amplification of the target regions of mecA and aac(6')/aph(2") that are responsible for the resistance to methicillin and arbekacin, respectively.