Genotyping of Streptococcus Pyogenes Isolates using Optimized RAPD-PCR Protocol

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Rahimi Moghaddam Akram; SALMANZADEH AHRABI SIAVOSH; FALSAFI TAHEREH; SEIFALI MAHVASH; Pourramezan Zahra

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Journal: Journal of Microbial Biology Year:2020 | Volume:8 | Issue:32 Page(s): 131-139

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Title

Genotyping of Streptococcus Pyogenes Isolates using Optimized RAPD-PCR Protocol

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Keywords

EpidemiologyQ1

Factorial DesignQ1

GenotypingQ1

RAPD-PCRQ2

Streptococcus PyogenesQ1

Abstract

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 protocol including 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 RAPDPCR 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.

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APA: Copy

Rahimi Moghaddam, Akram, SALMANZADEH AHRABI, SIAVOSH, FALSAFI, TAHEREH, SEIFALI, MAHVASH, & Pourramezan, Zahra. (2020). Genotyping of Streptococcus Pyogenes Isolates using Optimized RAPD-PCR Protocol. BIOLOGICAL JOURNAL OF MICROORGANISM, 8(32 ), 131-139. SID. https://sid.ir/paper/237333/en

Vancouver: Copy

Rahimi Moghaddam Akram, SALMANZADEH AHRABI SIAVOSH, FALSAFI TAHEREH, SEIFALI MAHVASH, Pourramezan Zahra. Genotyping of Streptococcus Pyogenes Isolates using Optimized RAPD-PCR Protocol. BIOLOGICAL JOURNAL OF MICROORGANISM[Internet]. 2020;8(32 ):131-139. Available from: https://sid.ir/paper/237333/en

IEEE: Copy

Akram Rahimi Moghaddam, SIAVOSH SALMANZADEH AHRABI, TAHEREH FALSAFI, MAHVASH SEIFALI, and Zahra Pourramezan, “Genotyping of Streptococcus Pyogenes Isolates using Optimized RAPD-PCR Protocol,” BIOLOGICAL JOURNAL OF MICROORGANISM, vol. 8, no. 32 , pp. 131–139, 2020, [Online]. Available: https://sid.ir/paper/237333/en

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