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

摘要 不同病原体的感染可以表现出类似的症状，但适当的治疗需要特定的 准确的诊断。临床医生经常转向多种微生物的多重检测（例如BioFire）。 虽然这些方法可以测试50-70个生物体，但它们不提供浓度滴度，这是不可能的。 有必要在几个假阳性中识别致病病原体或临床上无意义 - 谢谢因此，临床医生必须进行额外的测试，以确定哪些阳性是致病的。 尽管这些测试使用定量PCR，但在临床实验室中，由于以下原因，结果报告为存在/不存在 操作员认为，在这种情况下，PCR的挑剔性对反应效率的微小变化很敏感， 可变性因此，今天，只有少数广泛的PCR测试被FDA批准报告定量结果。 与qPCR相反，数字PCR（dPCR）通过计数单个分子来测量靶滴度。因此，在本发明中， dPCR提供不需要标准曲线的绝对浓度测量。此外该 反应循环至终点，然后定量;它不需要仔细估计扩增速率， 这是qPCR中变异性的主要来源。因此，dPCR对反应效率的变化不太敏感 并提供了优良的上级一致性。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个靶标，而qPCR可检测多达100个靶标。此外，dPCR需要复杂的微流体设备 这加重了测试实验室人员的负担并增加了成本。在这些问题得到解决之前，qPCR将继续进行。 控制临床实验室，定量和绝对的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的定量性和鲁棒性， qPCR的简单性和多重性。我们的愿景是实现广谱检测， 与高置信度定量滴度相关。我们的方法- gigapixel PCR（gPCR）-通过以下方式实现： 我们最近发现的自组装分区，用于无微流体产生单分散的 乳剂，和线性化的目标定量与毛细管电泳（CE）。CE允许灵敏定量 超过7个十年，并提供具有单核苷酸分辨率的扩增子长度信息。在gPCR中，我们用这个 以进行每个反应超过100个扩增子的多重检测。与qPCR相反，qPCR要求 样品被分开以测试不同的靶，从而稀释它并降低灵敏度，用gPCR测试靶 在不分裂的情况下进行测试，将它们保持在最大浓度，并显著增加灵敏度。 此外，基于稳健的dPCR，gPCR在测试条件下提供可重现的定量结果。它 从而解决了目前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， 与我们的长期合作者（Melanie Ott和Charles Chiu博士）合作，将其应用于呼吸和CNS 感染来自先前在UCSF医院收集的样本。

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.