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How do Coronavirus Disease (COVID-19) Tests Work?

Hartmut Pohl
Tags: Screening, Successful Research Tips



CDC/ Alissa Eckert, MS; Dan Higgins, MAMS

The COVID-19 pandemic is currently changing and challenging the world. The severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) causes the outbreak of this respiratory disease ranging from none or mild symptoms to severe pneumonia and life-threatening multi-organ failure. Reliable diagnostic tests that can detect acute or past infections with SARS-CoV-2 are a vital tool for healthcare professionals to diagnose COVID-19 early, monitor and treat patients. The test results are also a key metric for socio-political decisions to combat the spread of the pandemic. But how do these tests work?



Figure 1: Structure of the Virion and Genome of SARS-CoV-2, Wuhan-Hu-1 (RefSeq: NC_045512). SARS-CoV-2 is a enveloped, positive-sense, single-stranded ribonucleic acid (+ssRNA) virus. The virus genome contains 10 genes, some of which encode polyproteins which are split into multiple functional proteins after translation, resulting in a total of approximately 24-27 viral proteins. Four structural proteins form the viral capsid.
There are two main types of tests to detect COVID-19 in patients. The first type involves the detection of acute viral infection by detecting viral components. This is generally done by the detection of viral RNA genes with molecular genetic methods. The second type is serological testing using immunogenic methods to detect antibodies specific to SARS-CoV-2 proteins that the body produces in response to an infection.



Molecular Tests to Diagnose COVID-19

Molecular tests, which serve to detect genes of the virus in patients’ samples, are currently the dominant means to detect a coronavirus infection and diagnose COVID-19. These tests are generally categorized as Nucleic Acid Amplification Tests (NAATs) and all rely on reverse transcription (RT) of the viral RNA into DNA followed by polymerase chain reaction (PCR) amplification of the DNA and subsequent detection. Most frequently, the detection is based on reverse-transcription quantitative PCR (RT-qPCR). For more information on the technique, see our tech note about “What are the differences between PCR, RT-PCR, qPCR, and RT-qPCR?”.

The underlying basic principle (Figure 2) of these RT-qPCR-based tests is simple and always the same: specific genetic sequences within the viral genome are used to detect the virus specifically. For this purpose, a nasal or throat swab is performed to harvest viral particles and virus-infected mucosa cells. The swab sample is then lysed, the viral RNA extracted, and reverse-transcribed into cDNA. The cDNA in the processed sample is then quantified by qPCR to detect the presence of virus genome and confirm infection with the virus. While the principle is the same, the different test methods vary greatly in the single steps of the protocol. Automatization and optimization of sample preparation and reverse transcription or even performing it all in one reaction can reduce the time-to-results from several days to under an hour.

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Figure 2: Basic Principle of NAAT COVID-19 Test. Viral RNA is harvested and purified, followed by transcription into cDNA and subsequent amplification and detection by quantitative PCR.

Interestingly, not only the protocol may vary, but also which viral genes are detected. Most countries’ health care and disease control centers recommend the detection of 2-3 viral gene segments but which genes and sequences are detected vary greatly (Table 1). Procedures often rely on a normal human genetic target sequence as positive control. Additionally, pan-coronavirus probes might be used to detect the presence of various other coronaviruses alongside SARS-CoV-2.

Country Institute Targeted Genes Pan-CoV Probe
China China CDC ORF1ab, N
France Inst. Pasteur Paris ORF1ab(RdRP) (2 targets)
Germany Charité Berlin ORF1ab(RdRP), E, N Optional
Hong Kong SAR HKU ORF1ab(nsp14), N
Japan NIID Dept. Virol. II ORF1ab (multiple targets), S Yes
Thailand NIH N
USA CDC N (2 targets)

Table 1: Targeted Genes for SARS-CoV-2 testing by country (source WHO)

Besides RT-qPCR, whole viral genome sequencing by NGS is performed regularly on patient samples. While this method is not utilized as a diagnostic test, it occasionally serves to confirm the identity of SARS-CoV-2 in unclear test results and helps to monitor the geographic spread and genetic drift of the virus. Based on this monitoring, the PCR primers and targeted genes for RT-qPCR diagnostics may evolve over time.


Future Genetic Tests

One technique that could revolutionize yet another application, are CRISPR-based tests. These tests, which are currently in the proof-of-principle development stage use the CRISPR machinery that is capable of recognizing very specific target sequences and cutting them. In the process, a reporter construct is also cut which will then allow detection of the reaction. The key advantage is that this method would allow for the speed and ease of serology quick tests, with a test result within 5-10 minutes, but allow for the detection of viral genome markers and therefore would be able to be used in early diagnostics of the COVID-19 disease as a point-of-care test.



Immunogenic Detection to Diagnose COVID-19

An alternative method for detecting viral infection is the detection of blood-born viral antigens using antibody-based tests against viral proteins. However, this method faces severe obstacles. The concentration of viral antigens in typical samples currently in use, such as mucosa swabs, saliva or blood droplets, appears to be very low and successful detection of antigens by immunogenic methods may be challenging. Additionally, the detection of antigens by antibodies struggles to achieve competitive sensitivity to NAAT methods, which offer a far superior flexibility, as adjusting primer sequences is easily done.


Detecting the Immune Response in COVID-19

While genetic tests serve as the frontline diagnostic tool to monitor and combat the COVID-19 pandemic, immunogenic serology tests are used to detect antibodies that are being produced by the patient’s immune system in response to the virus infection. Due to the delay between infection, symptomatic onset and presence of antibodies, serological tests serve to diagnose past infections and assess a patient’s immune response rather than act as diagnostic tests to confirm an acute disease.

Antibodies, or immunoglobulins, exist as several classes. Immunoglobulin M (IgM) is released as a pentameric antibody and generally amongst the first responses of the humoral immune system. IgA is secreted as a dimeric antibody and plays a major role in the defense of the mucosal epithelia of the respiratory airways and the intestine. IgG typically appears later and forms the major component of the immune memory response and immunity. Several studies have indicated that SARS-CoV-2 might not follow this typical pattern though, and IgG antibodies against SARS-CoV-2 antigens arise in parallel with IgM and probably IgA responses. The detection of IgM without IgG appears to be uncommon. Seroconversion, i.e. the time period needed until specific antibodies are detectable in the blood, seems to often appear within several days after symptomatic onset, and serum antibody levels peak after 2-3 weeks. How long either IgM or IgG antibodies remain detectable following infection remains currently unknown.

The efficacy with which the adaptive immune system develops an antibody response and establishes lasting immunity following SARS-CoV-2 infection is currently debated. Laboratory studies and studies using samples from hospitalized patients suggest that nearly all immune competent individuals will develop an immune response against SARS-CoV-2. The results raise hope for a lasting immunity, as the genetic drift of SARS-CoV-2 has shown to be substantially slower than initially feared, and with ~8x104 nuclear substitutions per site per year, it is roughly one order of magnitude lower than for influenza virus. Additionally, crucial antigenic sites, such as the receptor binding domain (RBD) of the spike protein (aa 331-524), seem to be relatively conserved showing lower mutation rates. However, preliminary studies testing the general population indicate that minimal exposure to the virus or short disease courses with mild or no symptoms might not be sufficient to elicit a robust immune response. Additionally, many preliminary antibody screening studies have been hampered by the type and quality of the employed serological tests.


Serological Testing for Anti-SARS-CoV-2-Antibodies

Rapid detection of serologic antibodies against SARS-CoV-2 is often performed with point-of-care tests using a few blood drops from a finger prick. These tests are relative simple immunochromographic strip test (IST), often also called lateral flow test (LFT), and resemble in appearance to a common pregnancy test (Figure 3). They rely on immobilized antibodies and detection with colloidal gold-conjugated SARS-CoV-2 antigens. Colloidal gold is composed of very small gold particles (5-100nm), which will appear intense red. A few drops of blood, serum or plasma are added onto a sample pad and passed over a detection stripe by capillary tension. On its way, the sample passes a conjugate/reagent pad, where it is mixed with the conjugated viral antigens. When the blood contains antibodies that bind the viral antigen-conjugate, an antigen-antibody complex is formed. The sample-conjugate mix passes further over the detection stripes – zones where anti-human IgG/IgM antibodies have been spotted on. Here, the antibodies contained in the sample will be immobilized, and if they are bound to conjugated viral antigens, the detection stripe will be stained red. By having independent stripes of anti-IgM and anti-IgG antibodies, both subclasses of virus-specific antibodies can be detected individually. A positive control consisting of an antibody of a different species and the respective conjugate is included to indicate that the test was carried out correctly.

 


Figure 3: Basic Principle of Immunochromatic Strip Test (IST) Serological Tests. A blood sample is applied to the quick strip test. A buffer is applied to the reservoir and will distribute the sample through the test based on capillary forces. The sample passes a reagent reservoir, gets mixed with antigen-conjugate and IgM or IgG antibodies will be bound by capture antibodies in the detection stripe. If antigen-binding antibodies were present in the sample, the detection stripe will appear red.

Point-of-care lateral flow tests offer ease of use and rapid time-to-results, but IST is intrinsically hampered by the short incubation times defined by the capillary flow, the comparably small sample amounts, and the lack of wash steps, which limit the sensitivity and specificity of the assay type.

Enzyme-linked immunosorbent assays (ELISAs) overcome this issue and generally offer significantly better specificity and sensitivity at the cost of needing to be performed in a laboratory. Due to the microplate-based design of ELISAs, these tests can be easily automated and allow high-throughput screening of hundreds of patient samples at a time. Serological ELISA tests rely on very similar principles as lateral flow tests (Figure 4), most frequently with the roles of the antigen and detecting antibody reversed. The microplate is coated with SARS-CoV-2 antigens, with the antigen of choice most frequently being some variant of the spike protein. The spike protein (S) receptor binding domain (RBD) is thought to be amongst the most immunogenic epitopes on the viral capsid and is frequently used as an antigen of choice, amongst the S1 domain of the spike protein or full-length spike protein. Another antigen that initial data indicates to be highly immunogenic and especially play a major role in T cell responses is the nucleocapsid N. Due to its abundance, it is likely the antigen most frequently presented by infected cells, albeit not being detectable on intact viral particles. Therefore some ELISA tests use combinations of S and N to increase sensitivity at the cost of a slight reduction in specificity

 


Figure 4: Basic Principle of Enzyme-Linked Immunosorbent Assay (ELISA) Serological Tests. Antigen-coated microplates are used and a blood sample, most frequently plasma, is applied. The anti-SARS-CoV-2 antibodies contained in the blood sample will be immobilized by binding to the coated antigen. A detection conjugate, an enzyme-coupled secondary antibody, binds to the immobilized antibody and catalyzes a chromogenic reaction.

The sample –most often blood plasma or serum- is then added to the well, and SARS-COV-2-specific antibodies contained in the sample will bind to the antigen. An anti-human immunoglobulin antibody, which is conjugated to an enzyme, is added and the antibody-conjugate will bind to human antibodies bound to the antigen immobilized in the well. Most ELISAs detect either anti-SARS-CoV-2 IgG, IgM or IgA, requiring the use of distinct ELISA tests in parallel to detect all three subtypes of immunoglobulins. Wash steps in between every single binding step ensure that unbound or lightly, unspecific bound components do not interfere. A chromogenic substrate is used to quantify the amount of antibody-conjugate in a color reaction. The strength of the color reaction is directly proportional to the amount of bound antibody. This allows for a semi-quantitative interpretation of the antibody levels in a patient’s blood sample, but different binding affinities of the sample antibodies and antibodies contained in the controls impede a fully quantitative analysis. Nonetheless ELISA tests offer the possibility of some conclusions on the strength of the immune system’s reaction in addition to the improved sensitivity and specificity over IST. Also ELISA tests offer the ability to draw conclusions regarding the seroconversion of each immunoglobulin subclass.


Future Immunologic Tests

The field currently sees the emergence of multiplex ELISA assays that not only detect antibodies against several SARS-CoV-2 antigens but also allow the discrimination of antibodies against multiple antigen targets, as well as including negative and positive controls within the same well. Highly sophisticated multiplex automated blood immune assays combine detection of multiple antigen specificities with high throughput automation by sample dispersion in the nanoliter range, similar to flow cytometry, and allow the analysis of thousands of samples within a day. These tools might prove to be invaluable for large scale screenings and big clinical studies, but it is unlikely that they will become the diagnostic gold standard in the near future.



Enzo Life Sciences has over 40 years of expertise in genetics, labelling and detection. We encompass world-class experience in immunodetection with distinguished, top-of-the class immune assays. Furthermore, we offer a wide range of COVID-19-related products, from ready-to-use COVID-19 detection solutions, to qPCR reagents and antigens & antibodies, to useful tools and assays to study the biology of the virus. We even have you covered when it comes to small molecules and drug discovery, or even tools for upscaling your therapeutics production. For further insights into molecular biology or immunodetection, please have a look at our other Tech Notes. Or contact our Technical Support Team for further assistance.

 

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