Optimization of randomly amplified polymorphic DNA-polymerase chain reaction for molecular typing of Salmonella enterica serovar Typhi.

Bianca Ramalho Quintaes,Eliane Moura Falavina Reis,Ernesto Hofer,Nilma Cintra Leal

doi:10.1590/s0037-86822004000200006

Abstract

Optimization of the RAPD reaction for characterizing Salmonella enterica serovar Typhi strains was studied in order to ensure the reproducibility and the discriminatory power of this technique. Eight Salmonella serovar Typhi strains isolated from various regions in Brazil were examined for the fragment patterns produced using different concentrations of DNA template, primer, MgCl2 and Taq DNA polymerase. Using two different low stringency thermal cycle profiles, the RAPD fingerprints obtained were compared. A set of sixteen primers was evaluated for their ability to produce a high number of distinct fragments. We found that variations associated to all of the tested parameters modified the fingerprinting patterns. For the strains of Salmonella enterica serovar Typhi used in this experiment, we have defined a set of conditions for RAPD-PCR reaction, which result in a simple, fast and reproducible typing method.

Highlights

A otimização da reação de random amplification of polymorphic DNA fingerprinting technique (RAPD) para a caracterização de cepas de Salmonella enterica sorovar Typhi foi estudada com o objetivo de assegurar a reprodutibilidade e o poder discriminatório desta técnica
It has been demonstrated that RAPDPCR reaction has the potential to provide a discriminatory, reproducible and easy to interpret method to type S. enterica serovar Typhi strains
In order to use RAPD-polymerase chain reaction (PCR) for differentiation between bacterial strains, the optimization of the reaction is imperative to eliminate most of the variations that are sometimes observed in duplicate DNA profiles[24]

Summary

Introduction

A otimização da reação de RAPD para a caracterização de cepas de Salmonella enterica sorovar Typhi foi estudada com o objetivo de assegurar a reprodutibilidade e o poder discriminatório desta técnica. Para as amostras de Salmonella enterica sorovar Typhi utilizadas neste experimento, definiu-se um conjunto de condições para a reação de RAPD-PCR que resultou num método de tipagem simples, rápido e reprodutível. The amplification occurs at low stringency, allowing the primers to anneal to several locations on the two strands of the DNA. In order to use RAPD-PCR for differentiation between bacterial strains, the optimization of the reaction is imperative to eliminate most of the variations that are sometimes observed in duplicate DNA profiles[24]. In the present study we define the conditions for the optimization of RAPD-PCR to S. enterica serovar Typhi DNA using 10 bp primer and demonstrate the effects in the fingerprint pattern caused by varying the target DNA, MgCl and Taq DNA polymerase enzyme concentrations and the thermal cycling profile. We evaluate a total DNA extraction methodology, observing its time consumption and the stability of the resulting genetic material

Methods

Results

Conclusion