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

描述（由申请人提供）：本申请通过开发一种快速、简单的样本制备方法，解决了对沙眼衣原体（CT）扩大筛查和检测治疗计划的迫切需求。CT感染在女性患者中通常无症状，可导致感染个体的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 感染可以通过短疗程的抗生素很容易地解决，但是实现最佳的患者结果取决于在到医生办公室、医院或诊所的单次访问中识别感染并开出治疗处方--传染病护理的“测试和治疗”方法。 多项研究表明，目前的快速CT抗原检测显示出如此差的灵敏度，以至于在临床决策中几乎没有价值。相比之下，CT感染核酸检测的灵敏度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和低成本的增加，较小的诊所越来越适合在CT核酸检测中运行扩增和检测步骤。然而，用于随后的CT核酸检测的阴道拭子和尿液样本的必要准备-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。本申请寻求通过开发在廉价的一次性盒中的高性能、全自动CT样品制备来实现对CT核酸检测的扩展访问。使用过程包括简单地将尿液或阴道拭子样本放入一个与检测盒相连的小杯子中。狭缝毛细管阵列流体致动器（SCAFA）芯片--由申请人的公司最近开发的创新技术--被内置到一次性盒中，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、完全包埋集成（无离心）的固相提取方法，其在短玻璃毛细管内使用多孔聚合物整料，调节为具有最佳孔隙率以有效捕获核酸靶而不堵塞。在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、在过滤器上裂解CT和从过滤器洗脱的预提取步骤。试样入 到结果出来的时间是45分钟。第一阶段项目围绕两个具体目标进行组织。具体目标1侧重于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中的高产率提取，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，为第二阶段的全面开发奠定了基础。

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.