Matrix-based DNA Extraction for Targeted Next-Generation Sequencing on Decontaminated Sputum Samples.

IF 1.2 4区 综合性期刊 Q3 MULTIDISCIPLINARY SCIENCES
Jennifer Williams, Janré Steyn, Emilyn Costa Conceição, Felicia Bernita Wells, Melanie Grobbelaar, Nabila Ismail, Yonas Ghebrekristos, Christoffel Johannes Opperman, Sarishna Singh, Jason Limberis, Fahd Naufal, Brendon Coenrad Mann, Rebecca E Colman, Timothy Rodwell, Robin Mark Warren, John Z Metcalfe
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引用次数: 0

Abstract

Next-generation sequencing (NGS) is now recognized as a powerful tool for timely and accurate drug-resistant tuberculosis (DR-TB) diagnosis. Targeted NGS (tNGS) offers a streamlined approach by focusing on specific genes associated with drug resistance, bypassing the need for traditional culture-based methods with turnaround times ranging from weeks to months. The World Health Organization (WHO) has recommended tNGS as a valuable strategy for improving tuberculosis (TB) diagnosis to guide treatment and improve treatment outcomes, particularly in resource-limited settings. Among the WHO-recommended tNGS assays, we have selected a method that provides rapid and comprehensive drug susceptibility testing, lineage determination, and strain typing. While standardized DNA extraction methods are available, they can be time-consuming and labor-intensive. To address this challenge, we optimized a simplified, matrix-based DNA extraction protocol in combination with magnetic bead purification. This method offers a rapid and efficient approach for extracting DNA directly from decontaminated sputum sediments, enabling rapid downstream tNGS analysis. By streamlining the DNA extraction process from sputum sediment, this protocol could facilitate wider adoption of tNGS in routine clinical settings, ultimately contributing to improved patient outcomes and lending to global TB control efforts.

基于基质的DNA提取用于净化痰样本的下一代靶向测序。
下一代测序(NGS)现在被认为是及时和准确诊断耐药结核病(DR-TB)的有力工具。靶向NGS (tNGS)通过专注于与耐药性相关的特定基因提供了一种简化的方法,绕过了传统的基于培养的方法(周转时间从几周到几个月不等)的需要。世界卫生组织(世卫组织)建议将tNGS作为改进结核病诊断以指导治疗和改善治疗结果的一项宝贵战略,特别是在资源有限的环境中。在世卫组织推荐的tNGS检测方法中,我们选择了一种提供快速和全面的药敏试验、谱系测定和菌株分型的方法。虽然标准化的DNA提取方法是可用的,但它们可能既耗时又费力。为了应对这一挑战,我们优化了一种简化的、基于基质的DNA提取方案,并结合磁珠纯化。该方法提供了一种快速有效的方法,可以直接从去污染的痰沉积物中提取DNA,从而实现快速的下游tNGS分析。通过简化从痰液沉积物中提取DNA的过程,该方案可以促进在常规临床环境中更广泛地采用tNGS,最终有助于改善患者预后并促进全球结核病控制工作。
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来源期刊
Jove-Journal of Visualized Experiments
Jove-Journal of Visualized Experiments MULTIDISCIPLINARY SCIENCES-
CiteScore
2.10
自引率
0.00%
发文量
992
期刊介绍: JoVE, the Journal of Visualized Experiments, is the world''s first peer reviewed scientific video journal. Established in 2006, JoVE is devoted to publishing scientific research in a visual format to help researchers overcome two of the biggest challenges facing the scientific research community today; poor reproducibility and the time and labor intensive nature of learning new experimental techniques.
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