Determination of PCR-ELISA Diagnostic Value in Comparison With Classical Methods and PCR to Detect Resistance to Methacillin

Abstract

Background and Aims: High prevalence of Methicillin Resistant Staphylococcus Aureus isolates (MRSA) as well as the multi-drug resistance in this bacterium causes difficulties in the treatment of infections due to these bacteria. Hence, detection of MRSA isolates by rapid and accurate methods is necessary. PCR-ELISA is an accurate and molecular technique that is used for the detection of several pathogens. The aim of this study is the detection of MRSA using PCR-ELISA. Materials and Methods: Specific primers for mecA gene were designed. Then, dNTP labeled with Digoxigenin was applied for amplifying mecA gene. DIG-labeled PCR products were seeded on the well coated streptoavidin and identified by anti-DIG–peroxidase conjugate. Furthermore, Biotin-labeled DNA probe specific for mecA gene was used. Sensitivity and specificity of the method was determined. Resistance to methicillin among 70 clinical isolates was determined by the disk diffusion, agar dilution and PCR-ELISA methods. Results: MecA gene of S. aureus was amplified using gene specific primers resulted in a fragment with 310 bp length. Findings from the PCR-ELISA technic showed no cross-reactivity with Klebsiella Pneumoniae, Bacillus subtilis and Esheriashia coli as control bacteria and its sensitivity was 0.5 ng. The prevalence of MRSA clinical isolates by the disk diffusion, agar dilution and PCR-ELISA methods was 60%, 58.5% and 60%, respectively. Conclusion:The PCR-ELISA technique was known as an accurate and rapid test for the detection of infection agents using their specific gene. This technic can applied as an appropriate alternative method for time-consuming, less sensitive and expensive techniques such as Real-time PCR and differential biochemical tests which are currently used in laboratory.

Keywords

• staphylococcus aureus
• antibiotic resistance
• pcr-elisa
• meca gene