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1.
Biosens Bioelectron ; 166: 112455, 2020 Oct 15.
Article in English | MEDLINE | ID: covidwho-664093

ABSTRACT

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the coronavirus disease 2019 (COVID-19) worldwide pandemic. This unprecedented situation has garnered worldwide attention. An effective strategy for controlling the COVID-19 pandemic is to develop highly accurate methods for the rapid identification and isolation of SARS-CoV-2 infected patients. Many companies and institutes are therefore striving to develop effective methods for the rapid detection of SARS-CoV-2 ribonucleic acid (RNA), antibodies, antigens, and the virus. In this review, we summarize the structure of the SARS-CoV-2 virus, its genome and gene expression characteristics, and the current progression of SARS-CoV-2 RNA, antibodies, antigens, and virus detection. Further, we discuss the reasons for the observed false-negative and false-positive RNA and antibody detection results in practical clinical applications. Finally, we provide a review of the biosensors which hold promising potential for point-of-care detection of COVID-19 patients. This review thereby provides general guidelines for both scientists in the biosensing research community and for those in the biosensor industry to develop a highly sensitive and accurate point-of-care COVID-19 detection system, which would be of enormous benefit for controlling the current COVID-19 pandemic.


Subject(s)
Betacoronavirus/isolation & purification , Biosensing Techniques/methods , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Coronavirus Infections/virology , Pandemics , Pneumonia, Viral/diagnosis , Pneumonia, Viral/virology , Antibodies, Viral/isolation & purification , Antigens, Viral/isolation & purification , Betacoronavirus/genetics , Betacoronavirus/immunology , Biosensing Techniques/instrumentation , Biosensing Techniques/trends , Clinical Laboratory Techniques/instrumentation , Clinical Laboratory Techniques/trends , Coronavirus Infections/epidemiology , Equipment Design , Genome, Viral , Humans , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , Pneumonia, Viral/epidemiology , Point-of-Care Testing , RNA, Viral/genetics , RNA, Viral/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction , Virion/isolation & purification
2.
Sensors (Basel) ; 20(11)2020 May 31.
Article in English | MEDLINE | ID: covidwho-437172

ABSTRACT

One of the key challenges of the recent COVID-19 pandemic is the ability to accurately estimate the number of infected individuals, particularly asymptomatic and/or early-stage patients. We herewith report the proof-of-concept development of a biosensor able to detect the SARS-CoV-2 S1 spike protein expressed on the surface of the virus. The biosensor is based on membrane-engineered mammalian cells bearing the human chimeric spike S1 antibody. We demonstrate that the attachment of the protein to the membrane-bound antibodies resulted in a selective and considerable change in the cellular bioelectric properties measured by means of a Bioelectric Recognition Assay. The novel biosensor provided results in an ultra-rapid manner (3 min), with a detection limit of 1 fg/mL and a semi-linear range of response between 10 fg and 1 µg/mL. In addition, no cross-reactivity was observed against the SARS-CoV-2 nucleocapsid protein. Furthermore, the biosensor was configured as a ready-to-use platform, including a portable read-out device operated via smartphone/tablet. In this way, we demonstrate that the novel biosensor can be potentially applied for the mass screening of SARS-CoV-2 surface antigens without prior sample processing, therefore offering a possible solution for the timely monitoring and eventual control of the global coronavirus pandemic.


Subject(s)
Betacoronavirus/isolation & purification , Biosensing Techniques , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , Spike Glycoprotein, Coronavirus/isolation & purification , Antibodies, Viral/chemistry , Antibodies, Viral/immunology , Antigens, Viral/genetics , Antigens, Viral/isolation & purification , Betacoronavirus/pathogenicity , Coronavirus Infections/virology , Humans , Limit of Detection , Pandemics , Pneumonia, Viral/virology , Smartphone , Spike Glycoprotein, Coronavirus/chemistry
3.
Nature ; 583(7818): 834-838, 2020 07.
Article in English | MEDLINE | ID: covidwho-261141

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus with high nucleotide identity to SARS-CoV and to SARS-related coronaviruses that have been detected in horseshoe bats, has spread across the world and had a global effect on healthcare systems and economies1,2. A suitable small animal model is needed to support the development of vaccines and therapies. Here we report the pathogenesis and transmissibility of SARS-CoV-2 in golden (Syrian) hamsters (Mesocricetus auratus). Immunohistochemistry assay demonstrated the presence of viral antigens in nasal mucosa, bronchial epithelial cells and areas of lung consolidation on days 2 and 5 after inoculation with SARS-CoV-2, followed by rapid viral clearance and pneumocyte hyperplasia at 7 days after inoculation. We also found viral antigens in epithelial cells of the duodenum, and detected viral RNA in faeces. Notably, SARS-CoV-2 was transmitted efficiently from inoculated hamsters to naive hamsters by direct contact and via aerosols. Transmission via fomites in soiled cages was not as efficient. Although viral RNA was continuously detected in the nasal washes of inoculated hamsters for 14 days, the communicable period was short and correlated with the detection of infectious virus but not viral RNA. Inoculated and naturally infected hamsters showed apparent weight loss on days 6-7 post-inoculation or post-contact; all hamsters returned to their original weight within 14 days and developed neutralizing antibodies. Our results suggest that features associated with SARS-CoV-2 infection in golden hamsters resemble those found in humans with mild SARS-CoV-2 infections.


Subject(s)
Betacoronavirus/pathogenicity , Coronavirus Infections/transmission , Coronavirus Infections/virology , Disease Models, Animal , Lung/pathology , Lung/virology , Mesocricetus/virology , Pneumonia, Viral/transmission , Pneumonia, Viral/virology , Aerosols , Alveolar Epithelial Cells/pathology , Alveolar Epithelial Cells/virology , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antigens, Viral/immunology , Antigens, Viral/isolation & purification , Antigens, Viral/metabolism , Betacoronavirus/immunology , Betacoronavirus/isolation & purification , Betacoronavirus/metabolism , Bronchi/pathology , Bronchi/virology , Coronavirus Infections/immunology , Duodenum/virology , Fomites/virology , Housing, Animal , Kidney/virology , Male , Mesocricetus/immunology , Nasal Mucosa/virology , Pandemics , Pneumonia, Viral/immunology , RNA, Viral/analysis , Viral Load , Weight Loss
4.
JCI Insight ; 5(12)2020 06 18.
Article in English | MEDLINE | ID: covidwho-215032

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of human coronavirus disease 2019 (COVID-19), emerged in Wuhan, China, in December 2019. The virus rapidly spread globally, resulting in a public health crisis including almost 5 million cases and 323,256 deaths as of May 21, 2020. Here, we describe the identification and evaluation of commercially available reagents and assays for the molecular detection of SARS-CoV-2 in infected FFPE cell pellets. We identified a suitable rabbit polyclonal anti-SARS-CoV spike protein antibody and a mouse monoclonal anti-SARS-CoV nucleocapsid protein (NP) antibody for cross-detection of the respective SARS-CoV-2 proteins by IHC and immunofluorescence assay (IFA). Next, we established RNAscope in situ hybridization (ISH) to detect SARS-CoV-2 RNA. Furthermore, we established a multiplex FISH (mFISH) to detect positive-sense SARS-CoV-2 RNA and negative-sense SARS-CoV-2 RNA (a replicative intermediate indicating viral replication). Finally, we developed a dual staining assay using IHC and ISH to detect SARS-CoV-2 antigen and RNA in the same FFPE section. It is hoped that these reagents and assays will accelerate COVID-19 pathogenesis studies in humans and in COVID-19 animal models.


Subject(s)
Betacoronavirus/isolation & purification , Coronavirus Infections/virology , Pneumonia, Viral/virology , Animals , Antibodies, Viral/immunology , Antigens, Viral/isolation & purification , Betacoronavirus/genetics , Betacoronavirus/immunology , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Coronavirus Infections/pathology , Formaldehyde , Humans , Immunohistochemistry , In Situ Hybridization , Mice , Nucleocapsid Proteins/immunology , Pandemics , Paraffin Embedding/methods , Pathology, Molecular/methods , Pneumonia, Viral/pathology , RNA, Viral/isolation & purification , Rabbits , Tissue Fixation/methods
5.
Curr Protoc Microbiol ; 57(1): e100, 2020 06.
Article in English | MEDLINE | ID: covidwho-72322

ABSTRACT

In late 2019, cases of atypical pneumonia were detected in China. The etiological agent was quickly identified as a betacoronavirus (named SARS-CoV-2), which has since caused a pandemic. Several methods allowing for the specific detection of viral nucleic acids have been established, but these only allow detection of the virus during a short period of time, generally during acute infection. Serological assays are urgently needed to conduct serosurveys, to understand the antibody responses mounted in response to the virus, and to identify individuals who are potentially immune to re-infection. Here we describe a detailed protocol for expression of antigens derived from the spike protein of SARS-CoV-2 that can serve as a substrate for immunological assays, as well as a two-stage serological enzyme-linked immunosorbent assay (ELISA). These assays can be used for research studies and for testing in clinical laboratories. © 2020 The Authors. Basic Protocol 1: Mammalian cell transfection and protein purification Basic Protocol 2: A two-stage ELISA for high-throughput screening of human serum samples for antibodies binding to the spike protein of SARS-CoV-2.


Subject(s)
Antibodies, Viral/blood , Antigens, Viral/biosynthesis , Antigens, Viral/isolation & purification , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , Spike Glycoprotein, Coronavirus/biosynthesis , Spike Glycoprotein, Coronavirus/isolation & purification , Betacoronavirus/immunology , Coronavirus Infections/blood , Coronavirus Infections/immunology , HEK293 Cells , Humans , Pandemics , Pneumonia, Viral/blood , Pneumonia, Viral/immunology , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Seroconversion , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
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