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Bioessays ; 43(3): e2000257, 2021 03.
Article in English | MEDLINE | ID: covidwho-995863


Emergence of the novel pathogenic coronavirus SARS-CoV-2 and its rapid pandemic spread presents challenges that demand immediate attention. Here, we describe the development of a semi-quantitative high-content microscopy-based assay for detection of three major classes (IgG, IgA, and IgM) of SARS-CoV-2 specific antibodies in human samples. The possibility to detect antibodies against the entire viral proteome together with a robust semi-automated image analysis workflow resulted in specific, sensitive and unbiased assay that complements the portfolio of SARS-CoV-2 serological assays. Sensitive, specific and quantitative serological assays are urgently needed for a better understanding of humoral immune response against the virus as a basis for developing public health strategies to control viral spread. The procedure described here has been used for clinical studies and provides a general framework for the application of quantitative high-throughput microscopy to rapidly develop serological assays for emerging virus infections.

Antibodies, Viral/blood , COVID-19/diagnosis , Immunoassay , Immunoglobulin A/blood , Immunoglobulin G/blood , Immunoglobulin M/blood , Microscopy/methods , SARS-CoV-2/immunology , COVID-19/immunology , COVID-19/virology , COVID-19 Testing/methods , Fluorescent Antibody Technique , High-Throughput Screening Assays , Humans , Image Processing, Computer-Assisted/statistics & numerical data , Immune Sera/chemistry , Machine Learning , Sensitivity and Specificity
Viruses ; 12(8)2020 08 07.
Article in English | MEDLINE | ID: covidwho-713633


Rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The standard diagnostic pipeline for testing SARS-CoV-2 presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral RNA is extracted using commercial kits, followed by reverse transcription and quantitative PCR detection. As a result of the large demand for testing, commercial RNA extraction kits may be limited and, alternatively, non-commercial protocols are needed. Here, we provide a magnetic bead RNA extraction protocol that is predominantly based on in-house made reagents and is performed in 96-well plates supporting large-scale testing. Magnetic bead RNA extraction was benchmarked against the commercial QIAcube extraction platform. Comparable viral RNA detection sensitivity and specificity were obtained by fluorescent and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) using a primer set targeting the N gene, as well as RT-qPCR using a primer set targeting the E gene, showing that the RNA extraction protocol presented here can be combined with a variety of detection methods at high throughput. Importantly, the presented diagnostic workflow can be quickly set up in a laboratory without access to an automated pipetting robot.

Betacoronavirus/chemistry , Betacoronavirus/genetics , Clinical Laboratory Techniques/methods , Coronavirus Infections/virology , Pneumonia, Viral/virology , RNA, Viral/isolation & purification , Betacoronavirus/isolation & purification , COVID-19 , COVID-19 Testing , Coronavirus Infections/diagnosis , Humans , Magnetic Phenomena , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Pandemics , Pneumonia, Viral/diagnosis , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , Reverse Transcription , SARS-CoV-2 , Sensitivity and Specificity