Mycobacterium tuberculosis DNA Extraction Using InstaGene Matrix and High Speed Homogenizer from Clinical Primary Culture v1

Résumé

The conventional method for extracting Mycobacterium tuberculosis DNA, which involves the use of Cetyltrimethylammonium-bromide (CTAB) (Van Embden, 1993), is both labor-intensive and challenging to implement in routine diagnostic laboratory workflows. Shea et al. (2017) introduced an alternative approach for DNA extraction from clinical primary cultures (CPC) intended for Whole-Genome Sequencing (WGS). This alternative method utilizes the InstaGene Matrix (IGM) (Bio-Rad Laboratories, USA) which was modified and included the use of high-speed homogenization (HSH) through bead-beating such as FastPrep (FP) to improve efficiency and DNA yield. IGM is an optimized Chelex-100 resin designed to effectively bind metal ions such as Mg2+, which serves as essential cofactors in enzymatic reactions. IGM comprises 6% w/v Chelex chelating resin, incorporated during the 'pre-incubation' phase (at 56 °C for 30 minutes) to aid in the gentle disruption of plasma membranes and the disintegration of cell clumps, a task that cannot be accomplished solely with Chelex-100 chelating resin (Kang et al., 2018). In the context of Next-Generation Sequencing (NGS) techniques such as WGS or targeted NGS (tNGS), it is essential to incorporate a pre-treatment step before DNA extraction when working with CPC samples. This pre-treatment step is necessary to remove any potential contaminants that might be present in CPC samples that could adversely affect downstream sequencing analyses. For example, sequencing more of the contaminants' genomes than the target genome (WGS) or genes (tNGS) of M. tuberculosis. Initially, the IGM was primarily designed for solid culture sample use; thus, genomic DNA could be obtained from M. tuberculosis cultured on solid media such as 7H11 or Löwenstein–Jensen (LJ), as well as from liquid media such as 7H9, which includes the Mycobacteria Growth Indicator Tube (MGIT) (Becton Dickinson, Sparks, MD) media. This protocol describes the use of IGM/HSH for solid or liquid cultures and for WGS and/or tNGS applications as per Figure 1.

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