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

doi:10.4102/sajid.v40i1.717

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

S Afr J Infect Dis. 2025 May 27;40(1):717. doi: 10.4102/sajid.v40i1.717. eCollection 2025.

Authors

Christoffel J Opperman^{1

2

3

4}, Salim Ben Amor⁵, Greshan Kisten⁴, Brendon C Mann^{2

3}, Janré Steyn^{2

3}, Sarishna Singh^{2

3

4}, Yonas Ghebrekristos^{2

3

4}, Robin Warren^{2

3}, Wynand Goosen^{2

3

6}

Affiliations

¹ Department of Pathology, Faculty of Health Science, University of Cape Town, Cape Town, South Africa.
² Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa.
³ South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Stellenbosch University, Stellenbosch, South Africa.
⁴ National Health Laboratory Service, Green Point TB-Laboratory, Cape Town, South Africa.
⁵ Department of Medicine, Faculty of Medicine and Health Sciences, University of Cape Town, Cape Town, South Africa.
⁶ 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: DNA extraction; GenoLyse Version 1.0; Mycobacterium avium; Mycobacterium avium complex; amplicon-based sequencing; line probe assay; nontuberculous mycobacteria.