Rapid Identification of Methicillin-Resistant Staphylococcus mecA Gene by Polymerase Chain Reaction and Cloning of the Gene

ABSTRACT

The peneicillin binding protein gene(mecA gene) is present in the methicillin-resistant Staphylococcus strains but not in the susceptible ones. The goal of the present study was to establish experimental evidences which might use polymerase chain reaction(PCR) for culture confirmation and eventually clinical diagnosis of methicillin resistant Staphylococcui. Two primers (5'-AAAATCGATGGTAAAGGTTGGC-3', 5'-AGTTCTGCAGTACCGGATTTGC-3') based on the known DNA sequence of the mecA gene from methicillin-resistant Staphylococcus aureus were used in PCRs to screen for the presence of this gene in Staphylococcal isolates from various clinical settings. When the primers were used to copy the DNA of the mecA gene, only 533 base-pair DNA fragment was appeared. The product indicates a positive PCR result for methicillin-resistant Staphylococcal isolates. In contrast, from the DNA of the methicillin-sensitive Staphylococcal isolates the 533bp was not amplified. Results obtained with PCR were generally consistent with those of standard microbiological assays. The mecA gene in methicillin-high resistant Staphylococci was located on the approximately 5.56kb Hind III restriction fragment. The 533bp probe was hybridized to the 5.56kb Hind III restriction fragment of mecA-positive S. aureus. No hybridization was occured in the mecA-negative strain. The mecA gene was cloned, named pHL-1201 and verified by colony hyhridization. The 533bp probe was hybridized to the approximately 5.56kb Hind III restriction fragment of the DNA obtained from pHL-1201. PCRs with the primers successfully distinguished methicillin-resistants from methicillin-susceptible strains of S. aureus and S. epidermidis.