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Evaluating DNA extraction commercial kits from Mycobacterium tuberculosis clinical primary liquid (MGIT) culture for downstream sequencing applications

E C Conceicao(1) M Ismail(1) F Wells(1) B Mann(1) A Paulse(1) I Al-Talib(1) J Williams(1) A Dippennaar(1,2) G Van der Spuy(1) A Van Rie(2) R M Warren(1)

1:South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa; 2:Department of Family Medicine and Population Health, Global Health Institute, Faculty of Medicine and Health Sciences, University of Antwerp, Antwerp, Belgium

Whole-genome sequencing (WGS) has great potential for determining the complete drug-resistance profile of clinical Mycobacterium tuberculosis (Mtb) strains. Cetyltrimethylammonium-bromide (CTAB), the reference method for Mtb DNA extraction, is labour intensive and difficult to implement in routine laboratory settings. We evaluated the performance of commercial kits to extract DNA from clinical primary liquid cultures (CLPC). Mtb-positive decontaminated sputum sediments were pooled and used to inoculate mycobacteria-growth-indicator-tube (MGIT) 960 media. Positive non-contaminated MGIT cultures were pooled to generate 10 replicate isolates for DNA extraction by CTAB method with (+) or without (-) RNAse and seven commercial kits (+/-modifications): Zymo-DNA Clean and Concentrator, Zymo-Quick-DNA Fungal/Bacterial (+lysozyme-digestion), InstaGene (+/-bead-beat), GenoLyse (+/-precipitation), FluoroLyse (+/-precipitation), PrepGEM-Bacterial (+/-precipitation), NucleoSpin-Tissue, Nucleomag-Pathogen, and Gene-Xpert buffer (+precipitation or +purification). Genomic libraries for WGS were generated using Illumina DNA-Prep Kit and quality controlled using Qubit-dsDNA/HS and TapeStation. Nine performance parameters were evaluated: quantity of total dsDNA, DNA purity (Nanodrop spectrophotometry), DNA integrity through agarose-gel electrophoresis and PCR targeting the pncA gene (615 bp), quantity of genomic libraries (ng/uL), library fragment size (bp), turnaround time and cost. We developed a score for each performance parameter (1 to 5), used a radar-plot to visualize the scores and calculated the sum of scores for each method. The three best-performing methods were InstaGene +/-bead-beat (score=35), CTAB +RNAse (score=34), FluoroLyse +precipitation (score=33). The poorest performing kit was Zymo-Quick-DNA Fungal/Bacterial (score=11). Further evaluation by integrating WGS data quality scores will be performed to select the optimal commercially available kit(s) for routine downstream sequencing applications on CLPCs.

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