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

摘要 我们建议进行一项新的酶生物传感器技术的试验性测试， SARS-CoV-2的等温RNA扩增试验。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，以提高灵敏度，特异性， SARS-CoV-2检测速度。DETECT基于一种改良的分裂荧光素酶 互补分析代替标准的诱饵和猎物融合蛋白构建体，我们 将两个不相互作用的荧光素酶片段连接到SARS-CoV-2寡核苷酸探针上。 荧光素酶片段与寡核苷酸的偶联使用化学-酶促方法 在调查员的实验室里研发出来的寡核苷酸被设计成退火至相邻的寡核苷酸。 在一个独特的SARS-CoV-2扩增子的片段。对于含有扩增子的样品， 荧光素酶片段通过其附着的碱基配对聚集在一起 用SARS-CoV-2扩增子扩增寡核苷酸。分子组装重建功能 荧光素酶的两个片段，使鲁棒的光输出。在初步实验中，我们 验证DETECT的核心和新概念：通过以下方式实现蛋白质片段互补 核酸碱基配对。在寡核苷酸的碱基配对被阻断的对照中， 无论是通过核酸外切酶预处理或竞争寡核苷酸，我们观察到发光 读数与仅缓冲液样品相同。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- 200倍。 折叠背景。这些初步实验是用裂解的荧光素酶进行的， 寡核苷酸缀合物的浓度为25 nM。在这个为期两年的项目中，我们建议 定量评估DETECT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。虽然我们的目标是 诊断SARS-CoV-2感染，DETECT系统很容易通过改变 寡核苷酸探针序列。因此，DETECT有望成为一种新的创新 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.