Detecting COVID-19/SARS-CoV-2 Infections: Which test and when?

20 min Read

Rapid and robust diagnostic tests are essential to better manage and understand the spread of the SARS-CoV-2 virus. Currently, there are three types of tests available for COVID-19: Nucleic acid amplification tests, antigen diagnostic tests, and antibody detection tests. Nucleic acid amplification tests are the gold standard for the detection of active infections. Antigen and antibody tests are less sensitive but are more widely available due to their accessibility and affordability. Each test type has its own purpose:

  • Diagnostic tests are used to detect active coronavirus infections and determine whether quarantine or isolation is needed.
  • Antibody tests determine the long-term immune status of a previously-infected individual.

1. Nucleic Acid Amplification Tests (NAAT) Detect Active Infections

Real-time reverse transcription-polymerase chain reaction (RT-PCR) is a sensitive NAAT for diagnosing active SARS-CoV-2 infections. These tests are performed at laboratories, where trained technicians detect viral genetic material in human samples. First, RNA is extracted from a patient’s saliva or nasal swab. RNA is then reverse transcribed into cDNA by a reverse transcriptase enzyme and used as templates in PCR amplification. SARS-CoV-2 specific primers and probes have optimal sensitivity and specificity to the viral genetic sequence, allowing infections to be detected prior to symptom onset.

A typical RT-PCR workflow includes:

  • Patient sample collection (Nasal, Nasopharyngeal, Oropharyngeal, Sputum, Saliva)
  • RNA isolation from the patient sample.
  • Testing of the isolated RNA using a commercial RT-qPCR Detection Kit (kits may include COVID-19 primers and probes, reverse transcriptase, DNA polymerase, nucleotides, nuclease-free H2O, and reaction buffer).
  • The COVID-19 primers and probes included in the detection kit target SARS-CoV-2 proteins such as Rdrp, N protein, Orf1ab, and E protein, (read our article on SARS-CoV-2 structure and infection cycle).
  • The qPCR reaction is conducted via a thermocycler program that will heat and cool samples in a series of steps to carry out the denaturation, annealing, and elongation processes required to amplify and detect the target sequence.
  • The patient’s sample is run alongside positive and negative controls to ensure the run’s setup was done correctly and no sample contamination occurred, which would lead to false negatives and positives respectively. Detection of SARS-CoV-2 RNA indicates an active infection.
abm’s GenomeCoV19 Detection Kit (Cat. No. G628.v2) is a ready-to-use RT-qPCR test that includes all test components needed to detect SARS-CoV-2 in less than 2 hours. Our kit is affordably priced at US$1.77/test and is both widely used in Europe under the CE-IVD certification.

Figure 1 – Typical workflow for a SARS-CoV-2 RT-qPCR test.

2. Antigen Tests Detect Active Infections in Symptomatic Patients

Antigen tests are rapid immunoassays that detect viral protein in the patient’s nose or throat. Presence of SARS-CoV-2 nucleocapsid protein in nasopharyngeal and anterior nasal swab samples indicates an active infection. In comparison to RT-qPCR, immunoassay tests are cheaper, faster, and can be made available at a point of care location. Antigen tests are most effective at identifying the virus after symptoms have already developed, and may result in false negatives during early infections.

Similar to a rapid pregnancy test, many antigen tests for COVID-19 use a lateral flow test strip system and can take as little as 15 minutes to produce results.

  • Collect Nasopharyngeal and anterior nasal swabs from patient.
  • Release viral antigens using a commercial extraction buffer.
  • Apply sample extract to the disposable testing strip or testing cartridge.
  • Set a timer and wait as the sample is absorbed into the strip by capillary action, until it reaches the antibodies and detector molecules.
  • Interpret results based on manufacturer’s protocol.
3. Antibody Tests Detect Prior Infections and Immunity to COVID-19
Unlike RT-qPCR and antigen tests, antibody tests detect previous infections and active immunity against the virus. It may take up to 10 days post infection for a patient’s immune system to develop antibodies against COVID-19, making this test unsuitable as a diagnostics tool, but useful for determining a patient’s immune status.
An antibody test detects IgM and IgG antibodies in a patient’s blood samples for specificity against SARS-CoV-2 viral antigens. At 10 to 14 days post infection, the body produces an initial immune response and IgM antibodies may be detected. At 2 to 3 weeks post infection, IgG antibodies are released for long term immunity against the virus, and should be detectable long after the infection has been cleared.
The Enzyme-linked immunosorbent assay (ELISA) is a common antibody test that requires a microplate reader. It tests for the presence of anti-SARS-CoV-2 IgG in multiple patient samples at the same time. The following is a typical workflow:

  • Extraction and transport of blood samples to a laboratory for testing.
  • Isolate serum from blood samples.
  • Coat the wells of an ELISA plate with antigens recognized specifically by human anti-SARS-CoV-2 IgG antibodies.
  • Add blood serum to each well of the antigen coated plate.
  • Add anti-IgG secondary antibodies that are labelled with an enzyme that enables signal detection with an automated plate reader and quantitative analysis.
  • Incubate, then wash away unbound antibodies.
  • Interpret data according to the manufacturer's protocol.

Figure 2 – Typical IgG ELISA test workflow.

Alternatively, there are rapid test strips (such as the GenomeCoV19 IgM/IgG Test Kit) that detect anti-SARS-CoV-2 IgG and IgM antibodies in patient blood samples in less than 15 minutes. Inside each test strip is a conjugation pad containing SARS-CoV-2 recombinant antigens conjugated to colloidal gold for visualization, and a test region on the nitrocellulose membrane coated with anti-human IgM and IgG. If anti-SARS-CoV-2 antibodies are present in the sample, it will bind to the antigen conjugates and produce a visible dark band at the appropriate test line.

  • Use a transfer pipet to transfer 1 drop of whole blood, plasma, or serum to the sample well.
  • Add 2 to 3 drops of the kit’s buffer.
  • Wait 10 minutes for test results, follow test kit instructions for results interpretation.

Figure 3 – A typical lateral flow COVID-19 IgM/IgG antibody test strip. SARS-CoV-2 antibodies will bind to gold-antigen conjugates and be deposited along the IgM (M Line) or IgG (G Line) sites on the nitrocellulose membrane, appearing as a pink line to the naked eye.

4. Comparison between different methods of detecting SARS-CoV-2
Test Type Analyte Time to Results Use Case Detection Period Limitations
RT-qPCR Test Gene <2 hr Diagnoses active infections At any stage of an active infection (prior to symptom onset and up to 3 weeks after) Requires thermocycler and technician to perform test
Antigen Test Antigen <15 min Diagnoses active infections A few days after onset of symptoms Risk of false negatives due to dependence on amount of viral antigen produced
Antibody Test Antibody 1-3 days (ELISA-based test), <15 min (lateral flow strips) Immune status of previously infected patients 7-28 day after onset of symptoms Can not be used to detect active infections

Figure 4 – Chart of SARS-CoV-2 RNA, IgM and IgG levels during COVID-19 disease evolution.