Enabling point-of-care molecular diagnostics by developing an adaptive PCR instrument and on-demand kit reagents

聚合酶链反应（PCR）因其高灵敏度而成为诊断的金标准 许多传染病，但一般只在设备齐全的实验室实施。的一个主要 将PCR扩展到床旁市场的障碍是缺乏简单、稳健的单管PCR设计 其保持了其基于实验室的高灵敏度和特异性。在此快速通道STTR申请中， BioVentures公司和附近的研究机构范德比尔特大学提出， 不同的PCR设计，杂交触发PCR（HT-PCR）。它改变了PCR循环扩增的方式， 监测和控制，从而使设计更适合服务不足的护理点市场。HT-- PCR技术（由申请人共同发明）与BioVentures成功的商业模式非常吻合， 生产和销售分子生物学试剂，并与其扩展到临床的战略相一致 诊断市场。第一阶段和第二阶段的目标是评估这种方法的可行性和优势， 检测三种主要传染病的DNA生物标志物。 简单、稳健、单管PCR的主要障碍之一是有效扩增 反应需要窄范围的热和化学条件。护理点环境，包括步入式 诊所、农村卫生前哨和移动的反应单位的疫情监测，通常缺乏严格的 样品制备和受控的环境要求。的 当前所有PCR设计的基本限制是热循环由预定的控制 间接温度测量，但PCR产物解链步骤，更重要的是，引物 退火步骤并不总是发生在编程温度下。单个反应条件，环境 温度和热校准产生了预期的杂交状态之间的差异， 产物或引物和实际杂交状态。这些差异在诊断环境中加剧 它们不太能精确控制环境条件和样品含量，导致PCR 故障，即，假阴性我们提出了一种替代PCR设计，动态控制热循环 通过光学感测反应引物的镜像L-DNA替代物的退火和解链， 目标的因为L-DNA对映体的性质与天然D-DNA的性质相似，所以L-DNA试剂是 用于指示在每个过程中有效引物退火和产物熔化所需的循环条件 循环而不干扰反应。这种方法的一个主要优点是它能够实现杂交- 触发加热和冷却，而无需知道反应温度和时间。因此， 动态适应不可预测的热和化学变化。第二个主要优点是L- PCR产物的DNA替代物也可用作试剂再水化，样品制备， 仪器性能、诊断阈值和正确的产物形成，实现良好控制的单- DNA的试管分析

Because of its high sensitivity, polymerase chain reaction (PCR) is the gold standard for the diagnosis of many infectious diseases, but generally only implemented in well-equipped laboratories. One of the major roadblocks for expanding PCR to point-of-care markets is the lack of simple, robust, single tube PCR designs which preserve its laboratory-based high sensitivity and specificity. In this Fast-Track STTR application BioVentures, Inc. and Vanderbilt University, a nearby research institution, propose to develop a fundamentally different PCR design, hybridization-triggered PCR (HT-PCR). It alters the way PCR cyclic amplification is monitored and controlled, resulting in a design more suitable for underserved point-of-care markets. The HT- PCR technology (co-invented by the applicants) meshes well with BioVentures's successful business model of manufacturing and selling molecular biology reagents and aligns with its strategy to expand into the clinical diagnostic market. Phase I and II Aims evaluate the feasibility and advantages of this approach with studies to detect DNA biomarkers of three major infectious diseases. One of the major impediments to simple, robust, single tube PCR is that an efficient amplification reaction requires a narrow range of thermal and chemical conditions. Point-of-care settings, including walk-in clinics, rural health outposts, and outbreak surveillance by mobile response units, generally lack the stringent sample preparation and controlled environmental requirements available in centralized laboratories. The fundamental limitation with all current PCR designs is that thermal cycling is controlled by pre-determined indirect temperature measurements, yet the PCR product melting step and, more importantly, the primer annealing step, do not always occur at the programmed temperatures. Individual reaction conditions, ambient temperatures, and thermal calibrations create disparities between the expected hybridization state of the product or primers and the actual hybridization state. These disparities are exacerbated in diagnostic settings that are less equipped to precisely control environmental conditions and sample contents, leading to PCR failure, i.e., false negatives. We propose an alternative PCR design that dynamically controls thermal cycling by optically sensing the annealing and melting of mirror-image L-DNA surrogates of the reaction's primers and targets. Because the properties L-DNA enantiomers parallel those of natural D-DNAs, the L-DNA reagents are used to indicate the cycling conditions required for effective primer annealing and product melting during each cycle without interfering with the reaction. A major advantage of this approach is that it enables hybridization- triggered heating and cooling without the need to know reaction temperatures and times. Thus the instrument dynamically adapts to unpredictable thermal and chemical variations. A second major advantage is that the L- DNA surrogates of the PCR product can also be used as controls for reagent rehydration, sample preparation, instrument performance, diagnostic threshold, and correct product formation, enabling well-controlled single- tube analysis of DNA.