Next Generation Infectious Disease Diagnostics: Microfluidic-Free Gigapixel PCR with Self-Assembled Partitioning

摘要 不同病原体的感染可表现为相似的症状，但适当的治疗需要特定的 和准确的诊断。临床医生经常求助于多种生物检测方法(如BioFire)。 虽然这些方法可以检测50-70个微生物，但它们不提供浓度滴度，这是 有必要在几个假阳性或临床无意义的病例中确定致病原 共生关系。因此，临床医生必须进行额外的测试，以确定哪些阳性是致病的。 虽然这些测试使用定量聚合酶链式反应，但在临床实验室中，由于以下原因，结果被报告为存在/不存在 在这种情况下，聚合酶链式反应的挑剔性质，对反应效率的微小变化很敏感，操作员 可变性。因此，如今，只有少数几种广为流传的聚合酶链式反应检测方法被FDA批准用于报告定量结果。 与定量聚合酶链式反应不同，数字聚合酶链式反应(DPCR)通过计算单个分子来测量目标滴度。结果, DPCR提供了一种不需要标准曲线的绝对浓度测量方法。此外， 反应循环到终点，然后量化；它不需要仔细估计扩增速率， 这是定量聚合酶链式反应中变异的主要来源。因此，dpr对反应效率的变化不那么敏感。 并提供卓越的一致性。然而，目前的dPCR方法在多路复用方面受到限制，仅允许5- 每次检测6个靶点，而qPCR最多可以检测100个靶点。此外，直接聚合酶链式反应需要复杂的微流控设备 这给测试实验室人员带来了负担，并增加了成本。在这些问题得到解决之前，qpcr将继续下去。 主导临床实验室，定量和绝对病原体载量报告将仍然遥不可及。 在这里，我们提出了一种新的核酸技术，结合了dpcr的数量和稳健性与 定量聚合酶链式反应的简单性和多重性。我们的愿景是实现广谱检测，其中每个病原体 与高信任度、定量滴度有关。我们的方法--十亿像素聚合酶链式反应(GPCR)--由 我们最新发现的自组装分配，用于产生无微流体的单分散 乳剂，毛细管电泳线性靶标定量。行政长官允许灵敏的定量 超过70年，并提供单核苷酸分辨率的扩增片段长度信息。在gpcr中，我们使用这个 对每个反应进行超过100个扩增产物的多重检测。与qPCR相反，qPCR要求 样品被分开以检测不同的靶标，从而稀释它并降低灵敏度，用GPCR检测靶标 在没有分裂的情况下进行测试，将它们保持在最大浓度，并显著提高灵敏度。 此外，基于稳健的直接聚合酶链式反应，绿色聚合酶链式反应可在不同测试条件下提供可重复性的定量结果。它 从而解决了当前dPCR技术的主要局限性，并提供了第一个可行的替代方案 QPCR在临床上的应用。我们将根据公认的标准(SeraCare)开发和验证该技术，以及 与我们的长期合作伙伴(Melanie Ott和Charles Chiu博士)合作，将其应用于呼吸系统和中枢神经系统 来自以前在加州大学旧金山分校医院收集的样本的感染。

ABSTRACT Infections by different pathogens can manifest with similar symptoms, but appropriate treatment requires specific and accurate diagnosis. Clinicians often turn to multiplexed assays testing for many organisms (e.g. BioFire). While these approaches can test for 50-70 organisms, they do not provide concentration titers, which is necessary to identify the causative pathogen among the several false positives or clinically meaningless commensals. As a result, the clinician must perform additional tests to identify which of the positives is causative. Although these tests use quantitative PCR, in clinical labs the results are reported as presence/absence due to the finicky nature of PCR in this setting, which is sensitive to minor variations in reaction efficiency, operator variability. As a result, today, only a few widespread PCR tests are FDA approved to report quantitative result. In contrast to qPCR, digital PCR (dPCR) measures target titers by counting individual molecules. As a result, dPCR provides an absolute concentration measurement that doesn’t require a standard curve. In addition, the reaction is cycled to endpoint, then quantified; it does not require careful estimation of the amplification rate, which is a major source of variability in qPCR. Thus, dPCR is less sensitive to variations in reaction efficiency and provides superior consistency. However, current dPCR methods are limited in multiplexing, allowing just 5- 6 targets per assay, while qPCR can test up to 100. Moreover, dPCR requires complex microfluidic equipment that burdens testing lab personnel and increases cost. Until these issues can be addressed, qPCR will continue to dominate the clinical lab, and quantitative and absolute pathogen load reporting will remain beyond reach. Here, we propose a novel nucleic acid technology combining the quantitativeness and robustness of dPCR with the simplicity and multiplexing of qPCR. Our vision is to enable broad spectrum detection wherein each pathogen is associated with a high confidence, quantitative titer. Our approach – gigapixel PCR (gPCR) – is enabled by our recent discoveries of self-assembled partitioning, for microfluidic-free generation of monodispersed emulsions, and linearized target quantitation with capillary electrophoresis (CE). CE allows sensitive quantitation over 7 decades and provides amplicon length information with single nucleotide resolution. In gPCR, we use this to perform multiplexed detection of over 100 amplicons per reaction. In contrast to qPCR, which requires that the sample be split to test for different targets, thereby diluting it and reducing sensitivity, with gPCR the targets are tested without splitting, maintaining them at maximal concentration, and substantially increasing sensitivity. Moreover, based on robust dPCR, gPCR provides reproducible, quantitative results across testing conditions. It thus addresses the major limitations of current dPCR technologies and provides the first viable alternative to qPCR in the clinic. We will develop and validate the technology against accepted standards (SeraCare), and work with our longstanding collaborators (Drs. Melanie Ott and Charles Chiu) to apply it to respiratory and CNS infections from samples previously collected at UCSF hospitals.