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

Detection of Some Staphylococcal Enterotoxin Genes in MRSA Strains Using PCR Techniques

“…This study approaches to investigate toxin gene content by performing multiplex PCR technique (irrespective of whether the strain produces the toxin or not). A single PCR reaction (multiplex PCR amplification) was employed using five pairs of SEs (classical enterotoxins) following the general principles described by ( 15…”

“…Duplex PCR Fem A gene was used for molecular confirmation of Staph. aureus and mec A was used to detect the methicillin resistant strains directly without the need to proceed the step of antibiotic disc diffusion methods as referred by ( 14) following the procedure published by ( 15 ). The PCR cycling conditions were as follows: PCR reaction was conducted with (BiONEER.…”