Brief Report

Mycobacterium avium DNA extraction: Implications for NTM identification and amplicon sequencing

Christoffel J. Opperman, Salim Ben Amor, Greshan Kisten, Brendon C. Mann, Janré Steyn, Sarishna Singh, Yonas Ghebrekristos, Robin Warren, Wynand Goosen
Southern African Journal of Infectious Diseases | Vol 40, No 1 | a717 | DOI: https://doi.org/10.4102/sajid.v40i1.717 | © 2025 Christoffel J. Opperman, Salim Ben Amor, Greshan Kisten, Brendon C. Mann, Janré Steyn, Sarishna Singh, Yonas Ghebrekristos, Robin Warren, Wynand Goosen | This work is licensed under CC Attribution 4.0
Submitted: 22 January 2025 | Published: 27 May 2025

About the author(s)

Christoffel J. Opperman, Department of Pathology, Faculty of Health Science, University of Cape Town, Cape Town, South Africa; Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa; South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Stellenbosch University, Stellenbosch, South Africa; and National Health Laboratory Service, Green Point TB-Laboratory, Cape Town, South Africa
Salim Ben Amor, Department of Medicine, Faculty of Medicine and Health Sciences, University of Cape Town, Cape Town, South Africa
Greshan Kisten, National Health Laboratory Service, Green Point TB-Laboratory, Cape Town, South Africa
Brendon C. Mann, Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa; and South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Stellenbosch University, Stellenbosch, South Africa
Janré Steyn, Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa; South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Stellenbosch University, Stellenbosch, South Africa
Sarishna Singh, Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa; South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Stellenbosch University, Stellenbosch, South Africa; and National Health Laboratory Service, Green Point TB-Laboratory, Cape Town, South Africa
Yonas Ghebrekristos, Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa; South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Stellenbosch University, Stellenbosch, South Africa; National Health Laboratory Service, Green Point TB-Laboratory, Cape Town, South Africa
Robin Warren, Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa; South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Stellenbosch University, Stellenbosch, South Africa
Wynand Goosen, Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa; South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Stellenbosch University, Stellenbosch, South Africa; and Department of Microbiology and Biochemistry, Faculty of Natural and Agricultural Sciences, University of the Free State, Bloemfontein, South Africa

Abstract

This study evaluated six DNA (Deoxyribonucleic acid) extraction methods for Mycobacterium avium, focusing on cost, procedure time, yield, quality, DNA integrity, and suitability for amplicon-based sequencing. The extracted DNA was used in the GenoType® Mycobacterium Common Mycobacterium (CM) line probe assay (LPA), which is routinely used for nontuberculous mycobacteria (NTM) identification in South Africa.

Contribution: The findings demonstrate that the currently used DNA extraction method, GenoLyse® Version 1.0, remains the fastest, simplest, and most cost-effective approach for routine NTM identification, despite lower DNA yields. GenoLyse also shows potential for implementation in amplicon-based sequencing of NTM, specifically when amplifying the 16S rRNA gene.


Keywords

Mycobacterium avium; Mycobacterium avium complex; nontuberculous mycobacteria; DNA extraction; line probe assay; amplicon-based sequencing; GenoLyse Version 1.0

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