Facile Method for Enhanced Pathogen Detection

摘要 利用微流体技术进行高效和高精度的细胞样品处理，Tico Biosciences正在开发一种高效宿主细胞和宿主的免培养直接从痰液中分离的方法 从临床结核病（TB）样本中去除基因组DNA（gDNA），实现快速、简便和具有成本效益 全基因组测序（WGS）用于结核病药物敏感性诊断。耐药结核病是一个主要威胁 全球结核病控制，治疗和医疗干预战略在很大程度上依赖于有效的 诊断和筛查。然而，目前用于分枝杆菌样品处理和富集的方法， 结核病（Mtb）是严重不足。结核病诊断方法目前涉及直接来自 痰样本，导致宿主遗传物质的压倒性污染，或培养患者 样本，这是耗时的，并导致延迟的患者治疗过程和Mtb异质性的损失。 用于宿主细胞和宿主DNA去除的可用策略是劳动密集型的，具有高技能壁垒，并且导致 损失高达初始菌量的60%。因此，目前的技术水平导致患者的不良反应。 结果，特别是对于那些混合或耐药菌株。为了解决这些问题，Tico Biosciences已获得微流体技术的许可，该技术允许高效和高精度的细胞 样本处理，以去除直接来自痰液的无培养物样本中的宿主细胞和gDNA。这 方法将显著减少（1）总分析周转时间，（2）样品处理量，（3）用户技能 要求，（4）资源要求（如实验室空间、电力、标准生物实验室 设备等），以及（5）试剂和测序的每个样品成本为10倍。该方法导致 在快速去除超过99%的哺乳动物DNA，同时保留85%的混合细菌的初始载量方面， 加标人和E. coli细胞。在这个一期项目中，Tico计划进一步优化性能， 用模拟痰液对这种微流控系统进行了测试，并证明了这种方法对预存结核病的可行性 人类临床样品。这将通过以下方式实现：1）使用Tico的系统回收95%以上的细菌 直接从痰液样品中去除，同时去除超过99%的哺乳动物细胞， 显微镜和qPCR;以及2）证明Tico的系统能够直接从 痰液中少于10%的总读数映射到人类基因组。该项目将导致一个全面的 更快，更方便，更便宜的方法来处理临床痰液样本进行遗传分析， WGS或qPCR，并将转化为增加采用否则昂贵得令人望而却步的方法， 耐药性筛查，这是世界上许多地区迫切需要的。

Abstract Leveraging microfluidic technologies for high-efficiency and high-precision cellular sample processing, Tico Biosciences is developing a culture-free direct-from-sputum method for high-efficiency host cell and host genomic DNA (gDNA) removal from clinical tuberculosis (TB) samples, enabling rapid, facile, and cost-effective whole-genome sequencing (WGS) for TB drug susceptibility diagnostics. Drug-resistant TB is a primary threat to global TB control, and strategies for treatment and medical intervention are heavily dependent on effective diagnosis and screening. However, current methods for sample processing and enrichment of Mycobacterium tuberculosis (Mtb) are severely inadequate. TB diagnostic methods currently involve either WGS directly from sputum samples, resulting in the overwhelming contamination of host genetic material, or culturing of patient samples, which is time consuming and leads to delayed patient treatment courses and loss of Mtb heterogeneity. Available strategies for host cell and host DNA removal are labor-intensive, have high skill barriers, and result in loss of up to 60% of the initial bacillary load. Consequently, the current state of the art results in poor patient outcomes, especially for those with mixed or drug resistant strains. To address these limitations, Tico Biosciences has licensed microfluidic technologies that allow for high-efficiency and high-precision cellular sample processing in order to remove host cell and gDNA in culture-free direct-from-sputum samples. This method will significantly reduce (1) total analysis turn-around time, (2) amount of sample handling, (3) user skill requirements, (4) resource requirements (e.g. laboratory space, electrical power, standard biology lab equipment, etc.), and (5) per-sample costs for reagents and sequencing by a factor of ten. This method results in the rapid removal of over 99% of mammalian DNA while retaining 85% of the initial bacillary load from a mixed sample of spiked human and E. coli cells. In this Phase I project, Tico plans to further optimize the performance of this microfluidic system with mock sputum and demonstrate the feasibility of this approach on pre-banked TB human clinical samples. This will be accomplished by: 1) Using Tico's system to recover over 95% of bacteria directly from sputum samples while removing over 99% of mammalian cells as quantified by fluorescence microscopy and qPCR; and 2) Demonstrating Tico's system to enable culture-free WGS of Mtb directly from sputum with fewer than 10% of total reads mapped to the human genome. This project will lead to an overall faster, easier-to-use, and cheaper method of processing clinical sputum samples for genetic analyses such as WGS or qPCR and will translate to increased adoption of otherwise prohibitively expensive approaches for early drug resistance screening, which is critically needed in many regions around the world.