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

由于其高度敏感性，聚合酶链式反应(Pcr)是诊断的金标准。 在许多传染病中，但一般只在设备齐全的实验室实施。其中一个主要的 将聚合酶链式反应推广到医疗保健市场的障碍是缺乏简单、健壮、单管的聚合酶链式反应设计 从而保持了其基于实验室的高度敏感性和特异性。在此Fast-Track STTR应用程序中 BioVentures，Inc.和附近的研究机构范德比尔特大学(Vanderbilt University)提议开发一种根本 不同的聚合酶链式反应设计，杂交触发的聚合酶链式反应(HT-PCR)。它改变了PCR循环扩增的方式 监测和控制，从而设计出更适合服务不足的护理点市场。超级碗- 聚合酶链式反应技术(由申请者共同发明)与BioVentures成功的商业模式 制造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。第一和第二阶段的目的是评估这种方法的可行性和优势，并进行以下研究 检测三种主要传染病的DNA生物标志物。 简单、可靠、单管聚合酶链式反应的主要障碍之一是有效的扩增 反应需要很窄的热和化学条件。护理点设置，包括步入式 诊所、农村卫生前哨和移动应对单位的疫情监测通常缺乏严格的 在中央实验室提供样品制备和受控环境要求。这个 目前所有的聚合酶链式反应设计的基本限制是，热循环是由预先确定的 间接的温度测量，但PCR产物的融化步骤，更重要的是，引物 退火步骤中，不要总是在设定的温度下进行。单个反应条件，环境 温度和热校准在预期的杂交状态之间造成差异 产物或引物与实际杂交状态。这些差异在诊断环境中会加剧 设备较少，无法精确控制环境条件和样本含量，从而导致聚合酶链式反应 失败，即假阴性。我们提出了一种动态控制热循环的替代PCR设计 通过光学传感镜象L-DNA反应产物的退火和融化 目标。由于L脱氧核糖核酸对映体的性质与天然D-脱氧核糖核酸相似，因此L脱氧核糖核酸试剂是 用于指示有效的底漆退火所需的循环条件和在每个循环过程中的产品熔化 在不干扰反应的情况下循环。这种方法的一个主要优势是它能够进行杂交-- 无需知道反应温度和时间即可启动加热和冷却。因此，该仪器 动态适应不可预测的温度和化学变化。第二大优势是L-- 该聚合酶链式反应产物的DNA替代物也可用作试剂复水、样品制备、 仪器性能、诊断阈值和正确的产品形成，使良好控制的单 DNA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.