Mangalaparthi Kiran K, Chavan Sandip, Madugundu Anil K, Renuse Santosh, Vanderboom Patrick M, Maus Anthony D, Kemp Jennifer, Kipp Benjamin R, Grebe Stefan K, Singh Ravinder J, Pandey Akhilesh
Department of Laboratory Medicine and Pathology, Division of Clinical Biochemistry and Immunology, Mayo Clinic, Rochester, MN, 55905, USA.
Manipal Academy of Higher Education, Manipal, 576104, Karnataka, India.
Clin Proteomics. 2021 Oct 22;18(1):25. doi: 10.1186/s12014-021-09331-z.
SARS-CoV-2, a novel human coronavirus, has created a global disease burden infecting > 100 million humans in just over a year. RT-PCR is currently the predominant method of diagnosing this viral infection although a variety of tests to detect viral antigens have also been developed. In this study, we adopted a SISCAPA-based enrichment approach using anti-peptide antibodies generated against peptides from the nucleocapsid protein of SARS-CoV-2. We developed a targeted workflow in which nasopharyngeal swab samples were digested followed by enrichment of viral peptides using the anti-peptide antibodies and targeted parallel reaction monitoring (PRM) analysis using a high-resolution mass spectrometer. This workflow was applied to 41 RT-PCR-confirmed clinical SARS-CoV-2 positive nasopharyngeal swab samples and 30 negative samples. The workflow employed was highly specific as none of the target peptides were detected in negative samples. Further, the detected peptides showed a positive correlation with the viral loads as measured by RT-PCR Ct values. The SISCAPA-based platform described in the current study can serve as an alternative method for SARS-CoV-2 viral detection and can also be applied for detecting other microbial pathogens directly from clinical samples.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2），一种新型人类冠状病毒，在短短一年多时间里感染了超过1亿人，造成了全球疾病负担。逆转录聚合酶链反应（RT-PCR）是目前诊断这种病毒感染的主要方法，不过也已开发出多种检测病毒抗原的测试方法。在本研究中，我们采用了基于稳定同位素标准的细胞培养物抗体捕获法（SISCAPA）的富集方法，使用针对SARS-CoV-2核衣壳蛋白肽段产生的抗肽抗体。我们开发了一种靶向工作流程，其中对鼻咽拭子样本进行消化，然后使用抗肽抗体富集病毒肽段，并使用高分辨率质谱仪进行靶向平行反应监测（PRM）分析。该工作流程应用于41份经RT-PCR确认的临床SARS-CoV-2阳性鼻咽拭子样本和30份阴性样本。所采用的工作流程具有高度特异性，因为在阴性样本中未检测到任何目标肽段。此外，检测到的肽段与通过RT-PCR Ct值测量的病毒载量呈正相关。本研究中描述的基于SISCAPA的平台可作为SARS-CoV-2病毒检测的替代方法，也可直接用于从临床样本中检测其他微生物病原体。
