Why is qPCR considered the “gold standard” in COVID-19 testing?

qPCR- and antigen-based tests are two options for detecting SARS-CoV-2 infection. The qPCR test is considered the gold standard due to both its high sensitivity and specificity in SARS-CoV-2 detection. Sensitivity describes the test’s ability to correctly generate a positive result in individuals carrying the virus, while specificity describes the ability to correctly generate a negative result in individuals who are not infected. Both sensitivity and specificity are then reported as a percentage. The antigen test provides a quicker turnaround time but with variable sensitivity and specificity. Results from an antigen test should be confirmed with repeated testing or verification via qPCR. The qPCR test will continue to be the gold standard—especially as automation makes it possible to perform many qPCR-based tests quickly with minimal personnel.

How qPCR-based viral testing works1

qPCR-based tests detect the presence of viral RNA in a sample. The assay copies viral RNA into complementary DNA (cDNA) and then amplifies the cDNA using primers that are specific to genomic regions that encode for SARS-CoV-2 nucleocapsid proteins (N1 and N2) or RNase P.2  During amplification, the cDNA is quantified using fluorescent probes provided in the buffer. For example, the GeneCount® COVID-19 RT-qPCR Assay uses primers that target the genes encoding the N2 and envelope (E) proteins that are specific to SAR-CoV-2 and all SARS-Coronaviruses, respectively. The sensitivity and specificity of this assay was reported to be 100% and 97% (false positive and false negative), respectively. Furthermore, the GeneCount® COVID-19 RT-qPCR Assay provides a limit of detection (LoD) as low as 170 viral RNA copies/mL.

How antigen-based viral testing works

Unlike the qPCR-based tests, antigen-based tests are immunoassays that detect the presence of a viral antigen protein.3 These antigen tests therefore determine the presence of viral antigen rather than RNA in the collected sample. Antigen-based testing is both cheaper and quicker (15-30 minutes) than qPCR tests, which makes mass community testing possible. However, antigen tests are troubled with variable sensitivity and specificity that depends on whether an individual is symptomatic or who administers the test.

A case study in Arizona: sensitivity of antigen tests varies with symptoms4

The performance of the Abbott BinaxNOW Rapid Antigen Test was assessed at two testing sites in Arizona and was compared to RT-PCR tests and viral cultures. The sensitivity of the BinaxNOW tests varied depending on whether an individual was asymptomatic or symptomatic. Compared to RT-PCR tests, the sensitivity of the antigen test among asymptomatic individuals (35.8 %) was much less than symptomatic individuals (64.2%), but specificity remained high for both tests (99.8-100%). Among the specimens that were viral culture-positive, the sensitivity of the antigen test was much higher for both asymptomatic (78.6%) and symptomatic (92.6%) individuals. Thus, it can be inferred that the antigen test is often sufficient at detecting the presence of an active infectious virus. However, the sensitivity for positive specimen collected from asymptomatic individuals remains much lower, which suggests that these tests can miss those with lower viral load.

A case study in the UK: sensitivity of antigen tests varies depending on who performs them

The sensitivity of the antigen-based is also dependent on who performs them. The sensitivity of the Innova lateral flow antigen test among symptomatic individuals, when administered by laboratory scientists, was 79% but dropped to 58% when performed by self-trained staff.5 This variability in sensitivity was similarly observed with The Lateral Flow Viral Antigen detection devices (LFDs) as the false positive rate was significantly different based on whether the test was completed in the laboratory (0.06%) or field (0.39%).6

FDA & CDC recommend confirming positive antigen tests with qPCR

As a result, the FDA and CDC recommend following up with confirmatory qPCR tests for asymptomatic individuals whose antigen test comes back positive and for symptomatic individuals whose antigen test comes back negative.7,8 The CDC also states that “antigen tests perform best in symptomatic people and within a certain number of days since symptom onset,”3 which limits the accuracy of this test to a specific window of time.

The bottom line: gold standard for a reason

With the consideration that antigen-based testing provides quite variable sensitivity and specificity, it is no surprise that the FDA and CDC recommend follow-up qPCR testing. Even though antigen tests are useful in confirming SARS-CoV-2 infection in symptomatic individuals, asymptomatic cases still matter, as 40-45% of infected individuals are asymptomatic.9 In addition, as people are advised to isolate when feeling symptomatic, it is the asymptomatic individuals that pose a high risk in unknowingly spreading the SARS-CoV-2 virus. Therefore, testing should have high sensitivity among asymptomatic persons. The sensitivity of antigen tests could be increased to levels comparable to qPCR if testing multiple times per week10, but the speed and cost-effectiveness of antigen testing is negated if it must be repeated or confirmed with qPCR tests. To effectively limit the spread of SARS-CoV-2, it is also imperative that testing is consistently accurate regardless of the experience level of the person performing the tests. Although antigen tests provide a much quicker turnaround time, qPCR-based tests can be largely automated to not only improve turnaround time but also eliminate errors.


  1. Bustin, S. A. & Nolan, T. RT-qPCR Testing of SARS-CoV-2: A Primer. Int. J. Mol. Sci. 21, 3004 (2020).
  2. Real-time RT-PCR Primers and Probes for COVID-19 | CDC. https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-panel-primer-probes.html (2020).
  3. Interim Guidance for Antigen Testing for SARS-CoV-2 | CDC. https://www.cdc.gov/coronavirus/2019-ncov/lab/resources/antigen-tests-guidelines.html (2021).
  4. Prince-Guerra, J. L. Evaluation of Abbott BinaxNOW Rapid Antigen Test for SARS-CoV-2 Infection at Two Community-Based Testing Sites — Pima County, Arizona, November 3–17, 2020. MMWR. Morb. Mortal. Wkly. Rep. 70, 100–105 (2021).
  5. Grover, N. PCR, antigen and antibody: Five things to know about coronavirus tests. Horizon The EU Research & Innovation Magazine (2020).
  6. Preliminary report from the Joint PHE Porton Down & University of Oxford SARS-CoV-2 test development and validation cell: Rapid evaluation of Lateral Flow Viral Antigen detection devices (LFDs) for mass community testing. (2020).
  7. Potential for False Positive Results with Antigen Tests for Rapid Detection of SARS-CoV-2 – Letter to Clinical Laboratory Staff and Health Care Providers | FDA. https://www.fda.gov/medical-devices/letters-health-care-providers/potential-false-positive-results-antigen-tests-rapid-detection-sars-cov-2-letter-clinical-laboratory.
  8. SARS-CoV-2 Antigen Testing in Long Term Care Facilities | CDC. https://www.cdc.gov/coronavirus/2019-ncov/hcp/nursing-homes-antigen-testing.html (2021).
  9. Oran, D. P. & Topol, E. J. Prevalence of Asymptomatic SARS-CoV-2 Infection. https://doi.org/10.7326/M20-3012 173, 362–367 (2020).
  10. Smith, R. L. et al. Longitudinal Assessment of Diagnostic Test Performance Over the Course of Acute SARS-CoV-2 Infection. J. Infect. Dis. jiab337 (2021) doi:10.1093/INFDIS/JIAB337.

LuminUltra Team

Founded in 1995, LuminUltra is a molecular biology diagnostic testing company headquartered in Canada with operations in 6 countries. It is widely recognized globally as a leader in developing tests and reagents for environmental, industrial, and diagnostic monitoring applications and is a key supplier of COVID-19 diagnostic testing reagents to the Government of Canada.

Dozens of Fortune 500 customers across more than 80 countries trust LuminUltra’s technology, production reliability and history of customer service excellence to deliver their essential services in a safe state.

Related Posts

Your browser is out of date!

Update your browser to view this website correctly. Update my browser now