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

Southern African Journal of Infectious Diseases (May 2025)

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

Affiliations

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
Salim Ben Amor: Department of Medicine, Faculty of Medicine and Health Sciences, University of Cape Town, Cape Town
Greshan Kisten: National Health Laboratory Service, Green Point TB-Laboratory, Cape Town
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
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
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
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
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
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

DOI: https://doi.org/10.4102/sajid.v40i1.717
Journal volume & issue: Vol. 40, no. 1
pp. e1 – e5

Abstract

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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.

Published in Southern African Journal of Infectious Diseases

ISSN: 2312-0053 (Print); 2313-1810 (Online)
Publisher: AOSIS
Country of publisher: South Africa
LCC subjects: Medicine: Internal medicine: Infectious and parasitic diseases
Website: https://sajid.co.za

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