Genotyping of Streptococcus Pyogenes Isolates using Optimized RAPD-PCR Protocol

ژنوتايپينگ جدايه هاي استرپتوكوكوس پيوژنز با استفاده از پروتكل بهينه RAPD-PCR

Introduction: Streptococcus pyogenes causes a variety of infectious and non-infectious diseases. Typing of S. pyogenes isolates is one of the essential tools in the epidemiological studies of this bacterium. Random Amplified Polymorphic DNA (RAPD) is a rapid, easy and inexpensive PCR-based typing technique. Low reproducibility of RAPD-PCR is the main disadvantage of this method which will be resolved by optimization of RAPD-PCR protocol. Materials and methods: In this study, optimization of RAPD-PCR protocolincluding DNA extraction method, primer type, concentrations of PCR reagents, and PCR program was performed using the factorial design of experiments for S. pyogenes ATCC 19615 as a standard strain. Then, sixteen S. pyogenes isolates were genotyped by using optimized protocol. Typability, reproducibility, and discriminatory power of the optimized protocol were examined. Results: Among three DNA extraction methods and seven primers that were used, modified set buffer DNA extraction method and P14 primer were selected, respectively. Optimum concentration of PCR reagents were 3 mM MgCl2, 150 pmol primers, 0.2 mM dNTPs, 10 ng template DNA, and 2 U Taq DNA polymerase and the optimum PCR program consisted of an initial denaturation for 4 min at 94°C followed by 45 cycles of 1 min at 94°C, 2 min at 31°C, 2 min at 72°C, and a final extension at 72°C for 10 min.Results of optimized RAPD-PCR were reproducible for S. pyogenes ATCC 19615 and all S. pyogenes isolates. Calculated discriminatory power was satisfactory (DI=1). Sixteen S. pyogenes isolates belonged to sixteen strains which were classified into 3 main clusters on a similarity level of 14%. Discussion and conclusion: A suitable and reproducible RAPD-PCR protocol was obtained for genotyping of S. pyogenes isolates using RAPD-PCR optimization. The optimized protocol in the present study can be used in subsequent experiments on RAPD-PCR profiling for epidemiological study of S. pyogenes isolates.