Pdot-Enabled Point-of-Care Digital PCR for Sensitive Detection of SARS-CoV-2

项目摘要 目前SARS-CoV-2分子诊断试验大多是基于实时定量聚合酶链式反应 (QPCR)。数字聚合酶链式反应是基于限制稀释、终点的下一代聚合酶链式反应技术 聚合酶链式反应和泊松统计。数字聚合酶链式反应改变了定量聚合酶链式反应的指数性、模拟性 量化为线性、数字信号量化。与qPCR相比，数字聚合酶链式反应提供了 几个重要的优点，包括它提供绝对量化的能力，对 缓蚀剂/污染物及其高灵敏度。 最近的两项研究表明，数字聚合酶链式反应提高了检测新冠肺炎的敏感性 定量聚合酶链式反应没有成功。此外，对于一些患者，定量聚合酶链式反应对SARS-CoV-2感染的结果 每一天都不同。但与定量聚合酶链式反应不同的是，数字聚合酶链式反应具有一致的重复性。 总体而言，数字聚合酶链式反应被证明显著减少了假阴性结果，即 特别适用于早期或无症状感染的诊断或对恢复期的测试 患者出院前。 目前在定量聚合酶链式反应中使用的荧光探针，如FAM(一种荧光染料)，没有 使用手机类型的摄像头可以直观地显示亮度。要解决此问题并启用 简单的手机读取数字聚合酶链式反应的结果在护理点设置，该项目的目的是 将Pdots发展成可提供灵敏检测的数字聚合酶链式反应的超亮探针 SARS-CoV-2。

Project Summary Current SARS-CoV-2 molecular diagnosis tests mostly are based on real-time quantitative PCR (qPCR). Digital PCR is a next-generation PCR technology based on limiting dilution, end-point PCR, and Poisson statistics. Digital PCR transforms the exponential, analog nature of qPCR quantification into a linear, digital signal quantification. Compared to qPCR, digital PCR offers several important advantages, including its ability to provide absolute quantification, tolerance to inhibitors/contaminants, and its high sensitivity. Two recent studies showed digital PCR has improved sensitivity for picking up COVID-19 that qPCR had missed. In addition, for some patients, results on SARS-CoV-2 infections from qPCR varied from day to day. But unlike qPCR, digital PCR showed consistent reproducible results. Overall, digital PCR was shown to reduce significantly false-negative results, which is particularly useful for diagnosing early or asymptomatic infections or for testing convalescent patients before discharge. Current fluorescent probes used in qPCR, such as FAM (a fluorescein dye), do not have the brightness to be visualized with a cell-phone type camera. To address this issue and to enable simple cell-phone readout of digital PCR results in a point-of-care setting, this project aims to develop Pdots into ultrabright probes for digital PCR assays that can provide sensitive detection of SARS-CoV-2.