Evaluation of Simple and Cost-Effective DNA Preparation and Subsequent PCR Amplification for Clinically Relevant Mycobacteria
Veronica J. Gómez
Corporación Para Investigaciones Biológicas—(CIB), Bacteriology and Mycobacteria Unit, Carrera 72a N 78B-141, Medellín, Colombia and Universidad Pontificia Bolivariana—(UPB), Calle 78b No. 72a-109, Medellín, Colombia.
Angela M. Guzman
Corporación Para Investigaciones Biológicas—(CIB), Bacteriology and Mycobacteria Unit, Carrera 72a N 78B-141, Medellín, Colombia.
Gloria I. Mejia
Corporación Para Investigaciones Biológicas—(CIB), Bacteriology and Mycobacteria Unit, Carrera 72a N 78B-141, Medellín, Colombia.
Diego H. Caceres
Corporación Para Investigaciones Biológicas—(CIB), Bacteriology and Mycobacteria Unit, Carrera 72a N 78B-141, Medellín, Colombia.
Jaime A. Robledo
Corporación Para Investigaciones Biológicas—(CIB), Bacteriology and Mycobacteria Unit, Carrera 72a N 78B-141, Medellín, Colombia and Universidad Pontificia Bolivariana—(UPB), Calle 78b No. 72a-109, Medellín, Colombia.
Francois Rouzaud *
Corporación Para Investigaciones Biológicas—(CIB), Bacteriology and Mycobacteria Unit, Carrera 72a N 78B-141, Medellín, Colombia and Equal Opportunity Life Sciences, 1132 Parrish Dr., Rockville, MD, USA.
*Author to whom correspondence should be addressed.
Abstract
Aim: Slow growth rate in culture renders the traditional isolation, identification, and drug susceptibility testing of clinically important mycobacteria inadequate when there is an urgent need for a precise diagnosis in order to initiate patient treatment. Molecular methods all rely on mycobacterial DNA isolation which in turn has become an essential step of the process. Our study aimed to evaluate DNA isolation protocols from mycobacteria of clinical interest.
Methods: Therefore, in order to determine an optimal method we evaluated 8 inexpensive, rapid and easy DNA isolation methods from 30 mycobacterial cultures (10 Mycobacterium tuberculosis and 20 Non-tuberculous Mycobacteria) for subsequent direct detection by PCR.
Results: Six of those 8 methods reliably allow the isolation of good DNA yields and quality, the optimal protocol being the one that includes a 1% Triton X-100 lysis solution. Protocols using SDS 1% as a lysis solution did not yield DNA suitable for PCR amplification.
Conclusion: Six of the methods we evaluated can easily be implemented in resource limited settings for routine use, potentially contributing to a better management of mycobacterial infections.
Keywords: Mycobacteria, polymerase chain reaction, DNA isolation, diagnostics, resource limited settings