A novel cartridge-based sequencing solution for decentralized M. tuberculosis resistance detection

提出了一种新的分散M.结核病耐药性检测 耐利福平结核病（RR-TB）是与抗菌药物相关的主要死亡原因之一。 阻力目前在全球推荐的所有含贝达喹啉的口服RR-TB方案将缩短 治疗和改善结果。但由于缺乏及时的药敏数据增长缓慢 率M.结核病和需要高度封闭的生物实验室是扩大规模的主要障碍 贝达喹啉和其他新的和再利用的药物。Xpert MTB/RIF Ultra和其他市售分子TB 测试可以识别利福平耐药性，但不能告知完整的治疗方案，因为它们是有限的 仅分析少量遗传基因座（即，“热点”）。靶向下一代测序（NGS） 能够在不需要培养的情况下快速对整个基因进行测序，因此可以将RR-TB临床 管理然而，实现近患者临床应用有两个主要瓶颈：（1）复杂， 为NGS制备临床样本的非标准化工作流程，以及（2）成本和基础设施 符合行业标准Illumina平台的要求。与全球结核病诊断领导者Cepheid合作， 解决这些障碍，利用超声，微流体，和热循环能力的 现有的Xpert Ultra检测盒已在180多个国家使用， 系统与纳米孔基因测序平台的第一次。Xpert CartSeq，一种开创性的检测盒- 基于测序的解决方案，适用于较低水平的医疗保健，实现了稳健的DNA提取， 复杂的库准备，同时最大限度地减少用户的可变性和减轻技术技能要求。 通过本提案中详细介绍的开发，我们的目标是促进NGS在高血压患者中的临床应用。 负荷设置。

A novel cartridge-based sequencing solution for decentralized M. tuberculosis resistance detection Rifampin-resistant tuberculosis (RR-TB) is one of the principal causes of death associated with antimicrobial resistance. Newer all oral bedaquiline-containing RR-TB regimens, now recommended worldwide, will shorten treatment and improve outcomes. However, the lack of timely drug susceptibility data due to the slow growth rate of M. tuberculosis and the need for high containment biological laboratories are major barriers to scale-up of bedaquiline and other new and repurposed drugs. Xpert MTB/RIF Ultra and other commercial molecular TB tests can identify rifampin resistance, but cannot inform complete treatment regimens because they are limited to analyzing only a small number of genetic loci (i.e., “hot spots”). Targeted next-generation sequencing (NGS) is able to sequence entire genes rapidly without need for culture, and therefore could transform RR-TB clinical management. Achieving near-patient clinical application, however, has two major bottlenecks: (1) a complex, unstandardized workflow for preparing clinical samples for NGS, and (2) the cost and infrastructure requirements of industry-standard Illumina platforms. Partnering with global TB diagnostics leader Cepheid, we address these barriers by utilizing the ultrasonication, microfluidics, and thermocycler capabilities of the existing Xpert Ultra cartridge, already in use in over 180 countries, to unite a vetted direct-sample-to-answer system with a nanopore gene sequencing platform for the first time. Xpert CartSeq, a pioneering cartridge- based sequencing solution suitable for lower levels of healthcare, achieves robust DNA extraction and sophisticated library preparation while minimizing user variability and mitigating technical skill requirements. Through its development as detailed in this proposal, we aim to catalyze the clinical application of NGS in high burden settings.