Development of SYBR Green Real time PCR for identification methicillin-resistance Staphylococcus aureus (MRSA)

Abstract

The increasing resistance of staphylococci to ß -lactam antibiotics has become a major clinical problem. Development of rapid and sensitive techniques for detection of MRSA is an important aim for public health. A duplex PCR were established for specific identification of methicillin-resistance Staphylococcus aureus (MRSA) in clinical samples. In this work a duplex SYBR Green real time PCR was developed for rapid identification of MRSA in local methicillin-resistance S. aureus isolates. Twenty methicillin-resistance S. aureus isolates, as determined by disc diffusion method, were subjected to DNA extraction and PCR amplification. Two genes were amplified successfully, mecA (533bp) and femA (314bp), as targets for methicillin-resistance and specific identification of S. aureus, respectively using conventional PCR. Sensitivity of the duplex PCR showed that the minimum concentration of DNA that gave positive results for the two genes was 30ng/µl. In order to develop rapid and sensitive test for identification of MRSA, serial dilutions of purified DNA were amplified gradually according to their concentrations using SYBR Green real time PCR. These results indicated that the SYBR Green real time PCR can be used for identification of methicillin-resistance S. aureus (MRSA) in clinical