APHP, HUPSSD, Hôpital Avicenne, Service de Microbiologie Clinique, F-93009, Bobigny, France.
Université Sorbonne Paris Nord and Université Paris Cité, Inserm, IAME, F-93000, Bobigny, France.
Tuberculosis (Edinb). 2024 Sep;148:102543. doi: 10.1016/j.tube.2024.102543. Epub 2024 Jul 11.
The process of whole genome sequencing of the Mycobacterium tuberculosis complex is dependent on complete the inactivation of the strain and subsequent DNA extraction. The objective of this study was to optimise the two steps. Firstly, the efficacy of Triton X-100 as a solvent for the inactivation step was evaluated. This solvent has been demonstrated to be effective in killing bacteria and is less toxic than the previously employed chloroform. For the extraction step, two lysis methods were evaluated: enzymatic (B1 protocol) and mechanical (B2 protocol). For whole genome sequencing, the Nextera XT DNA library preparation protocol was performed for both the B1 and B2 protocols. Subsequently, each library was subjected to whole-genome sequencing. The results demonstrated that heat lysis inactivation with Triton was effective, with no bacteria remaining viable following this treatment. The enzymatic and mechanical extraction protocols yielded comparable results in terms of DNA quantity and quality. The sequencing results showed that there was no significant difference in read depths between the two protocols. In conclusion, for MTBC strains, we recommend the use of our Triton inactivation method, which meets biosafety expectations.
结核分枝杆菌复合体全基因组测序的过程依赖于菌株的完全失活和随后的 DNA 提取。本研究的目的是优化这两个步骤。首先，评估 Triton X-100 作为失活步骤溶剂的效果。这种溶剂已被证明能够有效地杀死细菌，而且比以前使用的氯仿毒性更小。对于提取步骤，评估了两种裂解方法：酶法（B1 方案）和机械法（B2 方案）。对于全基因组测序，对 B1 和 B2 方案都进行了 Nextera XT DNA 文库制备方案。随后，对每个文库进行全基因组测序。结果表明，用 Triton 进行热裂解失活是有效的，经过这种处理后没有细菌存活。酶法和机械法提取方案在 DNA 数量和质量方面产生了可比的结果。测序结果表明，两种方案之间的读取深度没有显著差异。总之，对于 MTBC 菌株，我们建议使用我们的 Triton 失活方法，该方法符合生物安全预期。
