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New Rapid Method for Differentiation of MRSA and SSA by PCR Restriction Analysis of 920 bp of dnaJgene
Zeinab H. Helal, Fatma Alzahraa M. Gomaa and Sahar M. R. Radwan
Microbiology and Immunology Department, Faculty of Pharmacy (Girls) Al-Azhar University, Nasr City, Cairo, Egypt. [email protected]
Abstract: Methicillin-resistant Staphylococcus aureus (MRSA) and sensitive Staphylococcus aureus(SSA) are responsible for a high proportion of nosocomial infections, which makes difficulty in treatment. MRSA infections are responsible for increased mortality rates, longer lengths of hospital stay, and higher rates of treatment failure compared to SSA infections. Detection of MRSA by conventional method is time consuming, influenced by culture medium, concentration of NaCl, temperature, time of incubation and antibiotics. Various PCR methods had been applied for the rapid detection and identification ofStaphylococcus species. The dnaJ gene sequence is potentially useful for the identification of genetically related Staphylococcus species and subspecies. While, with other bacterium PCR-restriction analysis is preferred, as a simple and cost-effective method that does not involve radioisotopes. For that reason in this study, we established and evaluated a rapid new protocol of identifying clinically relevant MRSA species by PCR- restriction analysis of the dnaJ gene. SSA and MRSA strains were isolated during a one year period from patients with bacterimia. Identification of S. aureus was performed by standard laboratory methods. Resistance to methicillin was detected by disc diffusion susceptibility test. DNA extraction was performed for both clinical blood samples as well as from isolated SSA and MRSA. Primers were designed to amplify specific dnaJ gene target and confirming the presence of S. aureus. MRSA was speciated by PCR-restriction analysis of dnaJ gene using XapI restriction enzyme. Our results showed two distinguished patterns of PCR-restriction analysis for SSA and MRSA. Only SSA is known to have a XapI restriction sites. Using our protocol, we were able to demonstrate the existence of staphylococcus and to identify their methicillin resistance. Therefore, we suggest that it would be very useful to apply PCR amplification restriction analysis to dnaJ gene directly to clinical specimens early in the diagnostic process. This would save several days that are required for conventional culture. Thus, this established protocol is suggested as a simple and useful method for the rapid detection and simultaneous identification of MRSA in primary clinical specimens or for the identification of culture isolates. This rapid detection would allow clinicians initially to avoid potentially inappropriate treatment options.
[Zeinab H. Helal, Fatma Alzahraa M. Gomaa and Sahar M. R. Radwan. New Rapid Method for Differentiation of MRSA and SSA by PCR Restriction Analysis of 920 bp of dnaJ gene. J Am Sci2012;8(12):832-837]. (ISSN: 1545-1003). http://www.jofamericanscience.org. 112
Keywords: MRSA, dnaJ gene, PCR Restriction Analysis. Full Text 112
