Lai Janice H, Keum Jung Won, Lee Haeun G, Molaei Mehdi, Blair Emily J, Li Sixing, Soliman Jesse W, Raol Vedant K, Barker Camille L, Fodor Stephen P A, Fan H Christina, Shum Eleen Y
bioRxiv. 2023 Oct 11:2023.10.09.561546. doi: 10.1101/2023.10.09.561546.
PCR has been a reliable and inexpensive method for nucleic acid detection in the past several decades. In particular, multiplex PCR is a powerful tool to analyze many biomarkers in the same reaction, thus maximizing detection sensitivity and reducing sample usage. However, balancing the amplification kinetics between amplicons and distinguishing them can be challenging, diminishing the broad adoption of high order multiplex PCR panels. Here, we present a new paradigm in PCR amplification and multiplexed detection using UltraPCR. UltraPCR utilizes a simple centrifugation workflow to split a PCR reaction into ∼34 million partitions, forming an optically clear pellet of spatially separated reaction compartments in a PCR tube. After thermocycling, light sheet scanning is used to produce a 3D reconstruction of the fluorescent positive compartments within the pellet. At typical sample DNA concentrations, the magnitude of partitions offered by UltraPCR dictate that the vast majority of target molecules occupy a compartment uniquely. This single molecule realm allows for isolated amplification events, thereby eliminating competition between different targets and generating unambiguous optical signals for detection. Using a 4-color optical setup, we demonstrate that we can incorporate 10 different fluorescent dyes in the same UltraPCR reaction. We further push multiplexing to an unprecedented level by combinatorial labeling with fluorescent dyes - referred to as "comboplex" technology. Using the same 4-color optical setup, we developed a 22-target comboplex panel that can detect all targets simultaneously at high precision. Collectively, UltraPCR has the potential to push PCR applications beyond what is currently available, enabling a new class of precision genomics assays.
在过去几十年里，聚合酶链反应（PCR）一直是一种可靠且经济的核酸检测方法。特别是多重PCR，它是一种强大的工具，能够在同一反应中分析多种生物标志物，从而最大限度地提高检测灵敏度并减少样本用量。然而，平衡扩增子之间的扩增动力学并区分它们可能具有挑战性，这限制了高阶多重PCR检测板的广泛应用。在此，我们展示了一种使用超PCR（UltraPCR）进行PCR扩增和多重检测的新范式。超PCR利用简单的离心流程将PCR反应分成约3400万个分区，在PCR管中形成由空间分离的反应隔室组成的光学透明沉淀。热循环后，使用光片扫描对沉淀内的荧光阳性隔室进行三维重建。在典型的样本DNA浓度下，超PCR提供的分区数量表明绝大多数目标分子都独特地占据一个隔室。这个单分子领域允许孤立的扩增事件发生，从而消除不同目标之间的竞争，并产生明确的光学信号用于检测。使用四色光学设置，我们证明可以在同一个超PCR反应中加入10种不同的荧光染料。我们通过荧光染料的组合标记（称为“复合技术”）将多重检测进一步提升到了前所未有的水平。使用相同的四色光学设置，我们开发了一个包含22个目标的复合检测板，能够高精度地同时检测所有目标。总体而言，超PCR有潜力将PCR应用拓展到目前的水平之上，实现一类新的精准基因组学检测。
