Diagnostic testing continues to be an integral component of the strategy to contain the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) global pandemic, the causative agent of Coronavirus Disease 2019 (COVID-19). The SARS-CoV-2 genome encodes the 3C-like protease (3CLpro) which is essential for coronavirus replication. This study adapts an in vitro colorimetric gold nanoparticle (AuNP) based protease assay to specifically detect the activity of SARS-CoV-2 3CLpro as a purified recombinant protein and as a cellular protein exogenously expressed in HEK293T human cells. We also demonstrate that the specific sensitivity of the assay for SARS-CoV-2 3CLpro can be improved by use of an optimised peptide substrate and through hybrid dimerisation with inactive 3CLpro mutant monomers. These findings highlight the potential for further development of the AuNP protease assay to detect SARS-CoV-2 3CLpro activity as a novel, accessible and cost-effective diagnostic test for SARS-CoV-2 infection at the point-of-care. Importantly, this versatile assay could also be easily adapted to detect specific protease activity associated with other viruses or diseases conditions.

中文翻译：

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开发用于检测 SARS-CoV-2 3CLpro 活性的比色法

诊断测试仍然是遏制严重急性呼吸系统综合症冠状病毒 2 (SARS-CoV-2) 全球大流行战略的一个组成部分，它是 2019 年冠状病毒病 (COVID-19) 的病原体。SARS-CoV-2 基因组编码对冠状病毒复制至关重要的 3C 样蛋白酶 (3CLpro)。本研究采用基于体外比色金纳米颗粒 (AuNP) 的蛋白酶测定法，专门检测 SARS-CoV-2 3CLpro 作为纯化重组蛋白和在 HEK293T 人类细胞中外源表达的细胞蛋白的活性。我们还证明，通过使用优化的肽底物和通过与无活性的 3CLpro 突变单体进行杂交二聚化，可以提高检测 SARS-CoV-2 3CLpro 的特异性灵敏度。这些发现突显了进一步开发 AuNP 蛋白酶检测的潜力，以检测 SARS-CoV-2 3CLpro 活性，作为一种新型、可访问且具有成本效益的即时诊断检测 SARS-CoV-2 感染。重要的是，这种多功能测定也可以很容易地适应检测与其他病毒或疾病状况相关的特定蛋白酶活性。