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.
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.
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
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.
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