Development of a colorimetric assay for the detection of SARS-CoV-2 3CLpro activity.
Development of a colorimetric assay for the detection of SARS-CoV-2 3CLpro activity.
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DOI：
10.1042/bcj20220105
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发表时间：
2022-04-29
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诊断检测仍然是遏制严重急性呼吸系统综合征冠状病毒2型（SARS-CoV-2）全球大流行战略的一个组成部分，该病毒是2019年冠状病毒病（COVID-19）的病原体。SARS-CoV-2基因组编码3C样蛋白酶（3CLpro），这是冠状病毒复制所必需的。本研究采用体外比色金纳米粒子（AuNP）为基础的蛋白酶测定，以特异性检测SARS-CoV-2 3CLpro的活性作为纯化的重组蛋白和作为外源表达在HEK 293 T人细胞的细胞蛋白。我们还证明，SARS-CoV-2 3CLpro检测的特异性灵敏度可以通过使用优化的肽底物和通过与无活性3CLpro突变单体的杂交二聚化来提高。这些研究结果强调了进一步开发AuNP蛋白酶测定法以检测SARS-CoV-2 3CLpro活性的潜力，该测定法作为一种新的、可获得的和具有成本效益的SARS-CoV-2感染的即时诊断试验。重要的是，这种多功能的检测方法也可以很容易地适用于检测与其他病毒或疾病相关的特定蛋白酶活性。
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.