year 13, Issue 1 (March - April 2019)
Iran J Med Microbiol 2019, 13(1): 22-31 |
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Susan Rezavand1 
, Jafar Amani 2, Neda Akbari1 , Hamid Reza Mohajerani1 , Shahram Nazarian3 , Hamideh Mahmoodzadeh Hosseini4 , Mehrdad Moosazadeh Moghaddam5
, Jafar Amani 2, Neda Akbari1 , Hamid Reza Mohajerani1 , Shahram Nazarian3 , Hamideh Mahmoodzadeh Hosseini4 , Mehrdad Moosazadeh Moghaddam5
1- Department of Biology, Faculty of Basic Sciences, Arak Branch, Islamic Azad University, Arak, Iran
2- Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran , jafar.amani@gmail.com
3- Department of Biology, Faculty of Basic Sciences, Imam Hussein University, Tehran, Iran
4- Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran
5- Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran
2- Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran , jafar.amani@gmail.com
3- Department of Biology, Faculty of Basic Sciences, Imam Hussein University, Tehran, Iran
4- Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran
5- Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran
Abstract: (6677 Views)
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.
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.
Type of Study: Original Research Article |
Subject:
Molecular Microbiology
Received: 2017/12/19 | Accepted: 2019/06/22 | ePublished: 2019/07/22
Received: 2017/12/19 | Accepted: 2019/06/22 | ePublished: 2019/07/22
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