McNerney Ruth, Clark Taane G, Campino Susana, Rodrigues Camilla, Dolinger David, Smith Liezel, Cabibbe Andrea M, Dheda Keertan, Schito Marco
Lung Infection and Immunity Unit, Department of Medicine, Division of Pulmonology and UCT Lung Institute, University of Cape Town, Old Main Building, Groote Schuur Hospital, Observatory, Cape Town, South Africa.
Faculty of Infectious and Tropical Diseases and Faculty of Epidemiology and Population Health, London School of Hygiene and Tropical Medicine, London, UK.
Int J Infect Dis. 2017 Mar;56:130-135. doi: 10.1016/j.ijid.2016.11.422. Epub 2016 Dec 13.
Whole genome sequencing (WGS) can provide a comprehensive analysis of Mycobacterium tuberculosis mutations that cause resistance to anti-tuberculosis drugs. With the deployment of bench-top sequencers and rapid analytical software, WGS is poised to become a useful tool to guide treatment. However, direct sequencing from clinical specimens to provide a full drug resistance profile remains a serious challenge. This article reviews current practices for extracting M. tuberculosis DNA and possible solutions for sampling sputum. Techniques under consideration include enzymatic digestion, physical disruption, chemical degradation, detergent solubilization, solvent extraction, ligand-coated magnetic beads, silica columns, and oligonucleotide pull-down baits. Selective amplification of genomic bacterial DNA in sputum prior to WGS may provide a solution, and differential lysis to reduce the levels of contaminating human DNA is also being explored. To remove this bottleneck and accelerate access to WGS for patients with suspected drug-resistant tuberculosis, it is suggested that a coordinated and collaborative approach be taken to more rapidly optimize, compare, and validate methodologies for sequencing from patient samples.
全基因组测序（WGS）能够对导致耐抗结核药物的结核分枝杆菌突变进行全面分析。随着台式测序仪和快速分析软件的应用，WGS有望成为指导治疗的有用工具。然而，直接从临床标本进行测序以提供完整的耐药谱仍然是一项严峻挑战。本文综述了当前提取结核分枝杆菌DNA的方法以及痰液采样的可能解决方案。正在考虑的技术包括酶消化、物理破碎、化学降解、去污剂增溶、溶剂萃取、配体包被磁珠、硅胶柱和寡核苷酸下拉诱饵。在WGS之前对痰液中的基因组细菌DNA进行选择性扩增可能提供一种解决方案，同时也在探索差异裂解以降低人类DNA污染水平。为消除这一瓶颈并加快疑似耐药结核病患者获得WGS检测的速度，建议采取协调合作的方法，以更快地优化、比较和验证从患者样本进行测序的方法。
