Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, King Abdulaziz University, P. O. Box 80402, Jeddah, 21589, Saudi Arabia.
Special Infectious Agent Unit, King Fahd Medical Research Centre, King Abdulaziz University, P. O. Box 80402, Jeddah, 21589, Saudi Arabia.
Mikrochim Acta. 2021 Mar 25;188(4):137. doi: 10.1007/s00604-021-04766-5.
The novel corona (SARS-CoV-2) virus causes a global pandemic, which motivates researchers to develop reliable and effective methods for screening and detection of SARS-CoV-2. Though there are several methods available for the diagnosis of SARS-CoV-2 such as RT-PCR and ELSIA, nevertheless, these methods are time-consuming and may not apply at the point of care. In this study, we have developed a specific, sensitive, quantitative and fast detection method for SARS-CoV-2 by fluorescence resonance energy transfer (FRET) assay. The total extracellular protease proteolytic activity from the virus has been used as the biomarker. The specific peptide sequences from the library of 115 dipeptides were identified via changes in the fluorescence signal. The fluorogenic dipeptide substrates have the fluorophore and a quencher at the N- and the C- terminals, respectively. When the protease hydrolyzes the peptide bond between the two specific amino acids, it leads to a significant increase in the fluorescence signals. The specific fluorogenic peptide (H-d) produces a high fluorescence signal. A calibration plot was obtained from the changes in the fluorescence intensity against the different concentrations of the viral protease. The lowest limit of detection of this method was 9.7 ± 3 pfu/mL. The cross-reactivity of the SARS-CoV-2-specific peptide was tested against the MERS-CoV which does not affect the fluorescence signal. A significant change in the fluorescence signal with patient samples indicates that this FRET-based assay might be applied for the diagnosis of SARS-CoV-2 patients. Graphical abstract.
新型冠状病毒（SARS-CoV-2）引发了全球性大流行，促使研究人员开发可靠、有效的 SARS-CoV-2 筛查和检测方法。虽然有几种方法可用于 SARS-CoV-2 的诊断，如 RT-PCR 和 ELSIA，但这些方法耗时且可能不适用于现场护理。在本研究中，我们通过荧光共振能量转移（FRET）测定法开发了一种针对 SARS-CoV-2 的特异性、灵敏性、定量和快速检测方法。将病毒的总细胞外蛋白酶的蛋白水解活性用作生物标志物。通过荧光信号的变化，从包含 115 个二肽的文库中鉴定出特定的肽序列。荧光二肽底物在 N 端和 C 端分别具有荧光团和猝灭剂。当蛋白酶水解两个特定氨基酸之间的肽键时，会导致荧光信号显著增加。特异性荧光肽（H-d）产生高荧光信号。通过荧光强度变化对不同浓度病毒蛋白酶的校准曲线获得。该方法的最低检测限为 9.7 ± 3 pfu/mL。针对 MERS-CoV 测试了 SARS-CoV-2 特异性肽的交叉反应性，其不影响荧光信号。与患者样本的荧光信号发生显著变化表明，这种基于 FRET 的测定方法可能适用于 SARS-CoV-2 患者的诊断。
