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1.
STAR Protoc ; 2(4): 100824, 2021 12 17.
Article in English | MEDLINE | ID: covidwho-1373303

ABSTRACT

For a cytopathic virus such as severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), the neutralization capacity of serum from convalescent or vaccinated persons or of therapeutic antibodies can be tested on adherent cell cultures. Here, a simple and tissue culture infectious dose-derived protocol for assessment of neutralization of SARS-CoV-2 is described. Compared with the often applied plaque-forming unit assay, the working load is lower, and fewer manipulations of the infected cultures are required. Hence, the method is safer for the personnel.


Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19/immunology , Neutralization Tests/methods , SARS-CoV-2/immunology , Viral Plaque Assay/methods , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/blood , COVID-19/therapy , Chlorocebus aethiops , Enzyme-Linked Immunosorbent Assay , Humans , Vero Cells
2.
J Med Virol ; 93(7): 4219-4241, 2021 07.
Article in English | MEDLINE | ID: covidwho-1151934

ABSTRACT

The potential zoonotic coronaviruses (SARS-CoV, MERS-CoV, and SARS-CoV-2) are of global health concerns. Early diagnosis is the milestone in their mitigation, control, and eradication. Many diagnostic techniques are showing great success and have many advantages, such as the rapid turnover of the results, high accuracy, and high specificity and sensitivity. However, some of these techniques have several pitfalls if samples were not collected, processed, and transported in the standard ways and if these techniques were not practiced with extreme caution and precision. This may lead to false-negative/positive results. This may affect the downstream management of the affected cases. These techniques require regular fine-tuning, upgrading, and optimization. The continuous evolution of new strains and viruses belong to the coronaviruses is hampering the success of many classical techniques. There are urgent needs for next generations of coronaviruses diagnostic assays that overcome these pitfalls. This new generation of diagnostic tests should be able to do simultaneous, multiplex, and high-throughput detection of various coronavirus in one reaction. Furthermore, the development of novel assays and techniques that enable the in situ detection of the virus on the environmental samples, especially air, water, and surfaces, should be given considerable attention in the future. These approaches will have a substantial positive impact on the mitigation and eradication of coronaviruses, including the current SARS-CoV-2 pandemic.


Subject(s)
COVID-19/diagnosis , High-Throughput Screening Assays/methods , Severe Acute Respiratory Syndrome/diagnosis , Enzyme-Linked Immunosorbent Assay/methods , Fluorescent Antibody Technique/methods , Genome, Viral/genetics , Humans , Middle East Respiratory Syndrome Coronavirus/genetics , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS Virus/genetics , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/genetics , Viral Plaque Assay/methods
3.
Diagn Microbiol Infect Dis ; 99(4): 115294, 2021 Apr.
Article in English | MEDLINE | ID: covidwho-987457

ABSTRACT

There remains an urgent need for assays to quantify humoral protective immunity to SARS-CoV-2 to understand the immune responses of COVID-19 patients, evaluate efficacy of vaccine candidates in clinical trials, and conduct large-scale epidemiological studies. The plaque-reduction neutralization test (PRNT) is the reference-standard for quantifying antibodies capable of neutralizing SARS-CoV-2. However, the PRNT is logistically demanding, time-consuming, and requires containment level-3 facilities to safely work with live virus. In contrast, a surrogate virus neutralization test (sVNT) manufactured by Genscript is a quick and simple assay that detects antibodies that inhibit the RBD-ACE2 interaction, crucial for virus entry into host cells. In this study, we evaluate the sensitivity, specificity, and cross-reactivity of the sVNT compared with the PRNT using both 50% and 90% SARS-CoV-2 neutralization as a reference-standard. We found that the sVNT provides a high-throughput screening tool prior to confirmatory PRNT testing for the evaluation of SARS-CoV-2 neutralizing antibodies.


Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Viral/blood , SARS-CoV-2/immunology , Viral Plaque Assay/methods , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/diagnosis , High-Throughput Screening Assays/methods , Humans , Neutralization Tests/methods
4.
Diagn Microbiol Infect Dis ; 99(2): 115248, 2021 Feb.
Article in English | MEDLINE | ID: covidwho-866639

ABSTRACT

As new tests and technologies advance our understanding and diagnostic capabilities of the severe acute respiratory syndrome coronavirus 2 and the coronavirus disease 2019, they must be appropriately validated to make sure test performance is following manufacturer claims. In this study, we evaluated the Vazyme 2019-nCoV IgG/IgM Detection Kit, which is a lateral flow assay (LFA), by the plaque reduction neutralization test (PRNT) using 100 patient plasma/serum samples. As compared to the PRNT results, the Vazyme LFA had 95.9% sensitivity and 96.1% specificity. Along with the increased need for rapid, effective, and affordable point of care tests to help provide meaningful epidemiological data, we demonstrated that the Vazyme LFA performed well on IgG detection but cannot be judged on the performance of IgM detection using PRNT alone. However, our observation of the low IgM-positive rate supported the poor performance of IgM detection of this LFA which led to the disapproval of its Emergency Use Authorization recently.


Subject(s)
Antibodies, Viral/blood , COVID-19/diagnosis , Neutralization Tests/methods , SARS-CoV-2/immunology , Viral Plaque Assay/methods , Humans , Immunoassay/methods , Immunoglobulin G/blood , Immunoglobulin M/blood , Point-of-Care Testing
5.
Nat Commun ; 11(1): 4812, 2020 09 23.
Article in English | MEDLINE | ID: covidwho-793542

ABSTRACT

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is commonly diagnosed by reverse transcription polymerase chain reaction (RT-PCR) to detect viral RNA in patient samples, but RNA extraction constitutes a major bottleneck in current testing. Methodological simplification could increase diagnostic availability and efficiency, benefitting patient care and infection control. Here, we describe methods circumventing RNA extraction in COVID-19 testing by performing RT-PCR directly on heat-inactivated or lysed samples. Our data, including benchmarking using 597 clinical patient samples and a standardised diagnostic system, demonstrate that direct RT-PCR is viable option to extraction-based tests. Using controlled amounts of active SARS-CoV-2, we confirm effectiveness of heat inactivation by plaque assay and evaluate various generic buffers as transport medium for direct RT-PCR. Significant savings in time and cost are achieved through RNA-extraction-free protocols that are directly compatible with established PCR-based testing pipelines. This could aid expansion of COVID-19 testing.


Subject(s)
Betacoronavirus/genetics , Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Coronavirus Infections/virology , Pneumonia, Viral/diagnosis , Pneumonia, Viral/virology , Reverse Transcriptase Polymerase Chain Reaction/methods , Benchmarking , COVID-19 , COVID-19 Testing , Clinical Laboratory Techniques/standards , Clinical Laboratory Techniques/statistics & numerical data , Coronavirus Infections/epidemiology , DNA Primers/genetics , Hot Temperature , Humans , Pandemics , Pneumonia, Viral/epidemiology , RNA, Viral/genetics , RNA, Viral/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction/standards , Reverse Transcriptase Polymerase Chain Reaction/statistics & numerical data , SARS-CoV-2 , Sensitivity and Specificity , Sweden/epidemiology , Viral Plaque Assay/methods
6.
Methods Mol Biol ; 2203: 135-143, 2020.
Article in English | MEDLINE | ID: covidwho-761350

ABSTRACT

Several techniques are currently available to quickly and accurately quantify the number of virus particles in a sample, taking advantage of advanced technologies improving old techniques or generating new ones, generally relying on partial detection methods or structural analysis. Therefore, characterization of virus infectivity in a sample is often essential, and classical virological methods are extremely powerful in providing accurate results even in an old-fashioned way. In this chapter, we describe in detail the techniques routinely used to estimate the number of viable infectious coronavirus particles in a given sample. All these techniques are serial dilution assays, also known as titrations or end-point dilution assays (EPDA).


Subject(s)
Coronavirus/pathogenicity , Viral Plaque Assay/methods , Animals , Cells, Cultured , Coronavirus/growth & development , Infectious bronchitis virus/growth & development , Infectious bronchitis virus/pathogenicity , Trachea/cytology
7.
Viruses ; 12(6)2020 06 06.
Article in English | MEDLINE | ID: covidwho-548042

ABSTRACT

In late 2019, a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, the capital of the Chinese province Hubei. Since then, SARS-CoV-2 has been responsible for a worldwide pandemic resulting in over 4 million infections and over 250,000 deaths. The pandemic has instigated widespread research related to SARS-CoV-2 and the disease that it causes, COVID-19. Research into this new virus will be facilitated by the availability of clearly described and effective procedures that enable the propagation and quantification of infectious virus. As work with the virus is recommended to be performed at biosafety level 3, validated methods to effectively inactivate the virus to enable the safe study of RNA, DNA, and protein from infected cells are also needed. Here, we report methods used to grow SARS-CoV-2 in multiple cell lines and to measure virus infectivity by plaque assay using either agarose or microcrystalline cellulose as an overlay as well as a SARS-CoV-2 specific focus forming assay. We also demonstrate effective inactivation by TRIzol, 10% neutral buffered formalin, beta propiolactone, and heat.


Subject(s)
Betacoronavirus/physiology , Coronavirus Infections/virology , Pneumonia, Viral/virology , Viral Plaque Assay/methods , Virus Inactivation , Animals , Betacoronavirus/drug effects , Betacoronavirus/growth & development , Betacoronavirus/pathogenicity , COVID-19 , Cellulose , Chlorocebus aethiops , Culture Media/chemistry , Formaldehyde , Guanidines/pharmacology , HEK293 Cells , Humans , Pandemics , Phenols/pharmacology , Propiolactone/pharmacology , SARS-CoV-2 , Sepharose , Vero Cells
8.
Curr Protoc Microbiol ; 57(1): ecpmc105, 2020 06.
Article in English | MEDLINE | ID: covidwho-437154

ABSTRACT

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been identified as the causal agent of COronaVIrus Disease-19 (COVID-19), an atypical pneumonia-like syndrome that emerged in December 2019. While SARS-CoV-2 titers can be measured by detection of viral nucleic acid, this method is unable to quantitate infectious virions. Measurement of infectious SARS-CoV-2 can be achieved by tissue culture infectious dose-50 (TCID50 ), which detects the presence or absence of cytopathic effect in cells infected with serial dilutions of a virus specimen. However, this method only provides a qualitative infectious virus titer. Plaque assays are a quantitative method of measuring infectious SARS-CoV-2 by quantifying the plaques formed in cell culture upon infection with serial dilutions of a virus specimen. As such, plaque assays remain the gold standard in quantifying concentrations of replication-competent lytic virions. Here, we describe two detailed plaque assay protocols to quantify infectious SARS-CoV-2 using different overlay and staining methods. Both methods have several advantages and disadvantages, which can be considered when choosing the procedure best suited for each laboratory. These assays can be used for several research purposes, including titration of virus stocks produced from infected cell supernatant and, with further optimization, quantification of SARS-CoV-2 in specimens collected from infected animals. © 2019 The Authors. Basic Protocol: SARS-CoV-2 plaque assay using a solid double overlay method Alternate Protocol: SARS-CoV-2 plaque assay using a liquid overlay and fixation-staining method.


Subject(s)
Betacoronavirus/isolation & purification , Clinical Protocols , Viral Plaque Assay/methods , Animals , Chlorocebus aethiops , Humans , SARS-CoV-2 , Staining and Labeling , Vero Cells
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