genotyping of streptococcus pyogenes isolates using optimized rapd-pcr protocol

Introduction: streptococcus pyogenes causes a variety of infectious and noninfectious 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 pcrbased 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.

Genotyping of Streptococcus Pyogenes Isolates using Optimized RAPD-PCR Protocol

Introduction: Streptococcus pyogenes causes a variety of infectious and noninfectious 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 PCRbased 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.