Comparison of four different DNA isolation methods from MGIT culture for long-read whole genome sequencing of Mycobacterium tuberculosis

authors:

avatar Nazlı ARSLAN ORCID 1 , * , avatar Ebru DEMIRAY-GURBUZ ORCID 2 , avatar Nuri OZKUTUK ORCID 3 , avatar Nuran ESEN ORCID 2 , avatar Ayse OZKUTUK ORCID 2

Department of Medical Microbiology, Institute of Health Sciences, Dokuz Eylül University, Izmir, Turkey
Department of Medical Microbiology, Faculty of Medicine, Dokuz Eylül University, Izmir, Turkey
Department of Medical Microbiology, Faculty of Medicine, Manisa Celal Bayar University, Manisa, Turkey
Jundishapur Journal of Microbiology: Vol.17, issue 9; e148070
article type: Research Article
revised: September 28, 2024

how to cite: ARSLAN N, DEMIRAY-GURBUZ E, OZKUTUK N, ESEN N, OZKUTUK A. Comparison of four different DNA isolation methods from MGIT culture for long-read whole genome sequencing of Mycobacterium tuberculosis. Jundishapur J Microbiol. 2024;17(9):e148070. 

Abstract

Background: Tuberculosis (TB) remains a global health challenge, particularly due to drug resistance and limitations in rapid diagnosis. Next-generation sequencing (NGS), especially long-read WGS, shows promise for rapidly detecting TB and drug resistance, but requires high-quality DNA, which is hard to extract from M. tuberculosis due to its complex cell wall. 
Objectives: This study evaluated four DNA isolation methods for extracting pure DNA from M. tuberculosis, aiming to standardize protocols for long-read WGS. 
Methods: M. tuberculosis H37RV colonies were grown in BACTEC MGIT liquid medium. Two pellets were prepared as the initial material for the DNA extraction protocol; pellets from 1ml McFarland 2 suspensions and all growing colonies from two MGIT liquid cultures. Four DNA extraction methods were used: the CTAB method and GeneJET-GenomicDNA-Purification Kit, Quick-DNA-Fecal/Soil-MicrobeKit, and Genematrix-Tissue/Bacterial-DNA-PurificationKit, with some modifications. DNA quality was assessed on the based on concentration, purity, and integrity. 
Results: Among the tested methods, the Quick-DNA-Fecal/Soil-MK, yielded approximately 85 ng/ml DNA yield and 1,9 purity at 260/280 nm from colonial pellet of two MGIT tubes. However, the lower intact DNA (DIN ̴ 6,8) was obtained by the kit. The CTAB method was provided the highest intact DNA (DIN ̴ 9.5) although the purity of DNA was not sufficient. 
Conclusions: Based on three repetitions of Mcf-2 and colonial pellet extractions, the Quick-DNA Fecal/Soil-MK kit yielded the highest DNA quantity and purity but showed lower integrity compared to other methods, indicating the need for adjustments. A pellet from two MGIT cultures (~100 µl) is suitable for long-read WGS with this kit. However, a larger sample size is required to generalize these findings. For effective long-read sequencing of M. tuberculosis, DNA extraction protocols must be optimized to balance yield, fragment size, and purity for accurate sequencing and drug resistance analysis.