Rapid No-Spin Chlamydia RNA Extraction via Filtering, Lysis and Monolith Binding

通过过滤、裂解和整体结合快速无旋转衣原体 RNA 提取

• 批准号: 8804242
• 负责人: Amy Droitcour
• 金额: $ 30万
• 依托单位: WAVE 80 BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-15 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8804242
• 关键词: Address; Adoption; Antibiotics; Antigens; Bacteria; Bacterial DNA; Bacterial RNA; Binding; Biological Assay; Blindness; Blood capillaries; Caring; Centrifugation; Chemistry; Chlamydia; Chlamydia trachomatis; Clinic; Clinical; Clinics and Hospitals; Communicable Diseases; Conjunctivitis; Cytolysis; DNA; Development; Diagnosis; Engineering; Environment; Exhibits; Female; Filtration; Flushing; Glass; HIV; Health; Individual; Infant; Infection; Infertility; Laboratories; Lead; Life; Methods; Mothers; Nucleic Acid Amplification Tests; Nucleic Acids; Outcome; Patients; Pelvic Inflammatory Disease; Performance; Phase; Polymers; Porosity; Positioning Attribute; Pregnant Women; Preparation; Procedures; Process; RNA; Reagent; Recovery; Reporting; Ribosomal RNA; Running; Sampling; Sensitivity and Specificity; Site; Solid; Staging; Swab; System; Technology; Test Result; Testing; Time; Urine; Vagina; Validation; Viral Load result; Visit; Woman; Work; amplification detection; capillary; clinical decision-making; cost; design; follow-up; high risk; improved; innovation; innovative technologies; instrument; manufacturing process; meter; programs; sample collection; screening; usability

DESCRIPTION (provided by applicant): This application addresses the urgent need for expanded screening and test-and-treat programs for Chlamydia trachomatis (CT) through the development of a fast, straightforward sample preparation method. CT infection, which is often asymptomatic in female patients, can result in pelvic inflammatory disease and irreversible infertility in the infected individual as well as blindness in infants born to infected mothers. CT infection can be readily resolved through a short course of antibiotics, but achieving optimal patient outcomes depends on identifying infection and prescribing treatment within a single visit to a doctor's office, hospital, or clinic-the "test-and-treat" approach to infectious disease care. Multiple studies have shown current rapid CT antigen tests to exhibit such poor sensitivity as to have almost no value in clinical decision making. In contrast, both the sensitivity and the specificity of nucleic acid testing for CT infection typically exceed 95%. With increasing availability and low cost of compact thermocyclers and other emerging nucleic acid testing technologies, smaller clinics are increasingly well positioned to run the amplification and detection steps in CT nucleic acid testing. Yet the requisite preparation of vaginal swab and urine samples for subsequent CT nucleic acid testing-including lysis, filtering, and extraction and elution of bacterial RNA and DNA-is a barrier to widespread adoption of CT nucleic acid testing outside of sophisticated laboratories. This application seeks to enable expanded access to CT nucleic acid testing through the development of high- performance, fully automated CT sample preparation in an inexpensive, disposable cartridge. The usage procedure consists of simply placing a urine or vaginal swab sample in a small cup, which attaches to the cartridge. Slit capillary array fluidic actuator (SCAFA) chips-an innovative technology recently developed by the Applicant Organization-are built into the disposable cartridge and drive all sample and reagent transport steps entailed in a high-extraction-efficiency sample preparation process. The extraction process will leverage an innovative, fully cartridge-integrated (no centrifugation) soli phase extraction method using porous polymer monoliths within short glass capillaries, tuned to have optimal porosity for efficient capture of a nucleic acid target without clogging. The sample preparation process executed within the cartridge also includes pre- extraction steps of sample metering, filtering CT from sample, lysing CT on the filter, and elution from the filter. Sample-in to results-out turnaround time is 45 minutes. This Phase I project is organized around two specific aims. Specific Aim 1 focuses on the sample prep module's front end, including metering and lysing. Specific Aim 2 focuses on high-yield extraction in the porous polymer monolith, including monolith composition and capture/elution chemistry. The two Aims collectively establish the feasibility of the high-performance, low-cost CT sample preparation cartridge, laying groundwork for full development in Phase II.

描述（由申请人提供）：本申请通过开发一种快速、简单的样品制备方法，解决了扩大沙眼衣原体 (CT) 筛查和测试与治疗计划的迫切需要。 CT 感染在女性患者中通常无症状，可导致感染者出现盆腔炎和不可逆的不孕症，以及感染母亲所生婴儿的失明。 CT 感染可以通过短期抗生素治疗轻松解决，但实现最佳患者治疗效果取决于在一次到医生办公室、医院或诊所就诊时识别感染并开出治疗处方，即传染病的“检测和治疗”方法关心。 多项研究表明，目前的快速 CT 抗原检测灵敏度较差，对临床决策几乎没有任何价值。相比之下，CT感染核酸检测的敏感性和特异性通常都超过95%。随着紧凑型热循环仪和其他新兴核酸检测技术的可用性不断提高且成本低廉，小型诊所越来越有能力运行 CT 核酸检测中的扩增和检测步骤。然而，后续 CT 核酸检测所需的阴道拭子和尿液样本的准备工作，包括裂解、过滤和提取， 细菌 RNA 和 DNA 的洗脱是在复杂实验室之外广泛采用 CT 核酸检测的障碍。该应用旨在通过在廉价的一次性试剂盒中开发高性能、全自动 CT 样品制备来扩大 CT 核酸检测的范围。使用过程包括简单地将尿液或阴道拭子样本放入连接到盒上的小杯子中。狭缝毛细管阵列流体致动器（SCAFA）芯片是申请人组织最近开发的一项创新技术，内置于一次性盒中，并驱动高提取效率样品制备过程中所需的所有样品和试剂运输步骤。提取过程将利用创新的、完全集成墨盒（无离心）的固相提取方法，该方法使用短玻璃毛细管内的多孔聚合物整体，调整为具有最佳孔隙率，可有效捕获核酸靶标而不会堵塞。在卡盒内执行的样品制备过程还包括样品计量、从样品中过滤CT、裂解过滤器上的CT以及从过滤器洗脱的预提取步骤。进样 得出结果的周转时间为 45 分钟。该第一阶段项目围绕两个具体目标进行组织。具体目标 1 重点关注样品制备模块的前端，包括计量和裂解。具体目标 2 侧重于多孔聚合物整料的高产率萃取，包括整料组成和捕获/洗脱化学。这两个目标共同确立了高性能、低成本CT样品制备盒的可行性，为二期的全面开发奠定了基础。