BACK

What do we know about PCR threshold cycles?

This article was published on
July 27, 2021

This explainer is more than 90 days old. Some of the information might be out of date or no longer relevant. Browse our homepage for up to date content or request information about a specific topic from our team of scientists.

Think about cycle thresholds like zooming in on a photo over and over again to find something small you’re looking for. If the thing you’re looking for in a photo is small like a mosquito, you’ll have to zoom in multiple times to see it. If it’s big and obvious like a firetruck, you won’t need to zoom in as much. Such is the case with PCR testing. The more viral particles in a test sample, the fewer cycles (or zoom ins) are required to detect the virus. Less viral material in a sample means more cycles are needed to find the small amounts of the virus.

Think about cycle thresholds like zooming in on a photo over and over again to find something small you’re looking for. If the thing you’re looking for in a photo is small like a mosquito, you’ll have to zoom in multiple times to see it. If it’s big and obvious like a firetruck, you won’t need to zoom in as much. Such is the case with PCR testing. The more viral particles in a test sample, the fewer cycles (or zoom ins) are required to detect the virus. Less viral material in a sample means more cycles are needed to find the small amounts of the virus.

Publication

What our experts say

PCR tests are the gold standard for detecting the COVID-19 virus. They are both reliable and accurate. Many types of PCR tests exist. For example, the University of Washington Laboratory offers about six different types. Sometimes, a laboratory can run more than one type of test to confirm a diagnosis.

All PCR tests currently follow the same basic principle. They try to match a piece of the DNA from a test swab (also called a sample) with the same genetic code from part of the SARS-COV-2 virus. 

The matching is key to determining if the virus is present. In order to detect the tiny DNA fragments, the PCR machine copies genetic fragments from the sample over and over again to zero in on key elements that confirm or deny a match with the SARS-COV-2 virus. This is a process called a cycle threshold. Each cycle doubles the number of viral RNA molecules from the sample exponentially so each cycle leads to many more of them.

Think about cycle thresholds like zooming in on a photo over and over again to find something small you’re looking for. If the thing you’re looking for in a photo is small like a mosquito, you’ll have to zoom in multiple times to see it. If it’s big and obvious like a firetruck, you won’t need to zoom in as much. Such is the case with PCR testing. The more viral particles in a test sample, the fewer cycles (or zoom ins) are required to detect the virus. Less viral material in a sample means more cycles are needed to find the small amounts of the virus.

The “cycle threshold” levels—the amount of zoom ins conducted to detect the virus—vary widely across regions and PCR machines. Several researchers from France, South America, and the United States looked at COVID-19 PCR test results from hospitalized patients. They found that the amount of cycles needed to detect the virus in the patients ranged between 14 and 45. 

Cycle thresholds are important for several reasons. Some types of PCR testing, called qualitative PCR tests, indicate whether a sample is positive or negative based on a limited number of cycles. If a manufacturer sets a cycling process to 30, the machine turns off after 30 rounds of copying and searching for SARS-CoV-2. If the machine detects the virus during those 30 rounds, it is reported as a positive test. If none is detected, it is reported as negative. 

A different type of PCR test called a quantitative PCR test does not have the same type of limit. These PCR tests help inform public health policies and management like contact tracing, or to help shape clinical treatment plans for patients. Once the quantitative PCR machine detects the viral DNA in the sample, it shuts off, regardless of which cycle it has completed. If it not does detect the viral DNA, it will stop after a certain number of cycles have been run. Quantitative PCR tests might be better at determining whether or not a person who was infected with COVID-19 is still infectious. 

Regulatory agencies like the United States Food and Drug Administration gave COVID-19 PCR test manufacturers a lot of flexibility in determining what the qualitative PCR test cycle threshold is to determine whether or not a sample is positive or negative. This is likely because these tests were approved through emergency use authorizations. Traditional, standardized cycle thresholds may not be realistic when fighting off a global pandemic. Many places still require that laboratories performing COVID-19 PCR tests report the cycle threshold numbers of both qualitative and quantitative tests.

There is currently some debate about how many times we should be copying and searching for the virus before determining whether a sample is positive or negative. Some opponents feel that the COVID-19 PCR test is run for too many cycles—in other words, gets copied too many times, thus making it an unreliable indicator of an infection. This group is pushing for the counts to be disclosed for each test so individual physicians, scientists, and lab technicians are able to determine whether or not a test is accurate. Most public health agencies have a requirement for cycle threshold counts for their respective populations, which informs local PCR testing protocols.

Ultimately, there is no standard cycle threshold value that is agreed upon internationally. The U.S. Food and Drug Administration currently gives laboratory manufacturers autonomy in determining how many cycles are needed to determine whether a sample is positive or negative. 

Regardless of the number of cycles that can be performed or ct-values, if there is no SARS-COV-2 viral genetic material from the starting sample to amplify, no virus is detected. If one is zooming in on a photo looking for a mosquito, and the mosquito isn’t there, then no amount of zooming in will reveal the bug. 

Context and background

There is an ongoing concern on different social media outlets about the usefulness of PCR tests in detecting SARS-COV-2 infections. Some ct-values mentioned by experts have been taken out of context or misunderstood.

Because of this, there has been some debate about how many threshold cycles must be run using PCR tests with different countries, regions, health agencies, and international bodies applying different rates.

There is also a challenge related to PCR tests’ ability to detect very small amounts of the virus. Even inactive viruses that are no longer infectious may show up as positive. The Lancet Microbe recently published a study which suggested that any live virus is no longer in the body nine days after symptoms first began. People are still able to shed the virus for weeks or months after this even if it’s not active or infectious. Thus, having a PCR test that detects viral genetic material doesn’t always mean a person is contagious. 

Despite these challenges, PCR tests are reliable even if the ct-values are high and are an excellent tool for diagnosing and detecting the SARS-CoV-2 virus in people.

Resources

  1. Rhoads, D., Peaper, D. R., She, R. C., Nolte, F. S., Wojewoda, C. M., Anderson, N. W., & Pritt, B. S. (2021). College of American Pathologists (CAP) Microbiology Committee perspective: caution must be used in interpreting the cycle threshold (Ct) value. Clinical Infectious Diseases, 72(10), e685-e686.
  2. Public Health Ontario
  3. University of Washington Medicine test guide
  4. Public Health Canada
  5. Mandatory Reporting of COVID-19 Laboratory Test Results: Reporting of Cycle Threshold Values (Florida Health Source)
  6. Engelmann, I., Alidjinou, E. K., Ogiez, J., Pagneux, Q., Miloudi, S., Benhalima, I., ... & Szunerits, S. (2021). Preanalytical issues and cycle threshold values in SARS-CoV-2 real-time RT-PCR testing: should test results include these?. ACS omega, 6(10), 6528-6536.
  7. AACC Recommendation for Reporting SARS-CoV-2 Cycle Threshold (CT) Values (AACC)
  8. Suklan, J., Cheaveau, J., Hill, S., Urwin, S. G., Green, K., Winter, A., Hicks, T., Boath, A. E., Kernohan, A., Price, D. A., Allen, A. J., Moloney, E., & Graziadio, S. (2021). Utility of Routine Laboratory Biomarkers to Detect COVID-19: A Systematic Review and Meta-Analysis. Viruses, 13(5), 803. (MDPI)
  9. RT-PCR for SARS-CoV-2: quantitative versus qualitative (The Lancet)
  10. An Overview of Cycle Threshold Values and their Role in SARS-CoV-2 Real-Time PCR Test Interpretation (Public Health Ontario
  11. Should qualitative RT-PCR be used to determine release from isolation of COVID-19 patients? (Journal of Infection
  12. Comparison of qualitative and quantitative analyses of COVID-19 clinical samples (Clinica Chimica Acta
  13. Explained: COVID-19 OCR Testing and Cycle Thresholds (Public Health Ontario
  14. Op-Ed: Why PCR Cycle Threshold Is Useful in Coronavirus Testing (MedPage Today

Media briefing

Media Release

Expert Comments: 

No items found.

Q&A

No items found.