Rapid SARS-CoV-2 Detection Using Amplicon Templated Reporter Enzyme Assembly

摘要 我们建议对一种新的酶生物传感器技术进行中试，以增强 SARS-CoV-2的恒温RNA扩增分析。我们的首要目标是验证这一点 技术，称为检测，作为生物分子工具，以提高敏感性，特异性，和 SARS-CoV-2检测的速度。检测是基于一种改进的分裂荧光素酶 互补试验。与标准的诱饵和猎物融合蛋白结构不同，我们 将两个非相互作用的荧光素酶片段连接到SARS-CoV-2寡核苷酸探针上。 利用化学酶方法将荧光素酶片段连接到寡核苷酸上 在调查员的实验室里研发出来的。寡核苷酸的设计是为了使相邻的 一种独特的SARS-CoV-2扩增片段。对于包含扩增子的样本，分裂 荧光素酶片段通过其连接的碱基配对而聚集在一起 带有SARS-CoV-2扩增子的寡核苷酸。分子组装重组功能 来自两个片段的荧光素酶，使强健的光输出。在初步实验中，我们 验证检测的核心和新概念：通过 核酸碱基配对。在阻止寡核苷酸碱基配对的对照中， 无论是核酸外切酶预处理还是竞争寡核苷酸处理，我们都观察到了发光 读数与仅缓冲样本相当。相比之下，在实验样本中， 支持寡核苷酸碱基配对，我们观察到荧光素酶信号增加了100- 在背景上折叠。这些初步实验是用分裂的荧光素酶- 寡核苷酸在25 nm处结合。在这个为期两年的项目过程中，我们建议 定量评估检测系统的特异性、灵敏度和速度，从而 评估这种生物传感器技术的临床潜力。尽管我们在这里的目标是 诊断SARS-CoV-2感染，通过改变检测系统很容易重新编程 寡核苷酸探针序列。因此，Detect作为一种新的、创新的 诊断平台。

ABSTRACT We are proposing to pilot test a new enzyme biosensor technology for the purpose of enhancing isothermal RNA amplification assays for SARS-CoV-2. Our overarching goal is to validate this technology, called DETECT, as biomolecular tool to increase the sensitivity, specificity, and speed of SARS-CoV-2 testing. DETECT is based on a modified split luciferase enzyme complementation assay. Instead of the standard bait and prey fused protein constructs, we connect two non-interacting luciferase fragments to SARS-CoV-2 oligonucleotide probes. Conjugation of the luciferase fragments to the oligonucleotides uses a chemi-enzymatic method developed in the investigator's lab. The oligonucleotides are designed to anneal to adjacent segments in a unique SARS-CoV-2 amplicon. With samples containing the amplicon, the split luciferase fragments are brought together through base pairing of their attached oligonucleotides with the SARS-CoV-2 amplicon. Molecular assembly reconstitutes functional luciferase from the two fragments, enabling robust light output. In preliminary experiments, we validate the central and novel concept of DETECT: protein fragment complementation via nucleic acid base pairing. In controls where base pairing of the oligonucleotides is blocked, either by exonuclease pretreatment or by competitor oligonucleotide, we observe luminescence readings on par with buffer only samples. By contrast, in experimental samples where oligonucleotide base pairing is supported, we observe luciferase signal that is increased 100- fold over background. These preliminary experiments were carried out with the split luciferase- oligonucleotide conjugates at 25 nM. Over the course of this 2-year project, we propose to evaluate the DETECT system quantitatively for specificity, sensitivity and speed, thereby assessing the clinical potential of this biosensor technology. Although our objective here is diagnosing SARS-CoV-2 infection, the DETECT system is easily re-programmed by changing the oligonucleotide probe sequences. Thus DETECT holds promise as a new and innovative diagnostic platform.