Explained: COVID-19 PCR Testing and Cycle Thresholds


17 Feb 2021

PCR, DNA, RNA, sensitivity, amplification, cycle thresholds? 

There’s a lot of information out there on COVID-19 testing and you’ve probably come across these words before. But what do they mean? Why are they important for COVID-19 testing? And how do they all fit together? We are going to break it all down for you. 

Understanding COVID-19 terms

Before we start, it’s important to understand some common terms. 

Coronavirus: are a large family of viruses that cause disease in mammals and birds. In humans, they are known to cause respiratory illness such as the common cold, which is mostly caused by seasonal coronaviruses. Novel coronaviruses include the viruses that cause Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS).

SARS-COV-2: is the name of the specific coronavirus responsible for the COVID-19 pandemic.

COVID-19: is the disease caused by SARS-CoV-2 virus.

Back to Basics: DNA and RNA

Let’s go back to science class — remember all those lessons on DNA (deoxyribonucleic acid) and RNA (ribonucleic acid)? DNA and RNA are genetic material found in living things, including humans, animals, plants – and even viruses. They carry the specific blue print or building blocks for how living things are made and developed (the genetic code). The genetic blueprint in humans is coded in DNA. Viruses are different as most viruses either have DNA or RNA (not both). The genetic code for SARS-CoV-2 (which we will refer to from now on as the virus) is coded in RNA
So, why do we need to know this? Well, the COVID-19 lab test is actually looking for the genetic material of the virus (we’ll get to that soon).

PCR Testing: The Gold Standard

PCR testing (also known as polymerase chain reaction testing) was developed over 35 years ago and is one of the most widely used lab tests for finding viruses (and other pathogens like bacteria, fungi and parasites) that cause diseases such as Ebola, SARS and now COVID-19 (SARS-CoV-2). The World Health Organization recommends Nucleic Acid Amplification Testing (which includes PCR testing) as the preferred testing method for COVID-19 and tells us if someone is infected with the virus. 

"In Ontario, we use PCR as the gold standard of testing for COVID-19 because it is able to successfully detect tiny amounts of the virus (sensitivity) with a low chance for error (accuracy) compared to other types of lab tests."

— Jonathan Gubbay, Medical Microbiologist, Public Health Ontario

The test requires a sample from a person, which is collected by a health care provider. The gold standard for sample collection method is the nasopharyngeal swab, a swab inserted deep into a person’s nose. However, other sample types exist including combinations of a nose and throat swab and also saliva samples.

Now that you have a bit of background information, let’s get into some of the most common questions people ask about PCR testing.

How does PCR actually detect COVID-19?

As we mentioned earlier, the PCR tests are designed to look for the virus’ genetic material. Since coronaviruses don’t have DNA, the first step of PCR testing is converting the virus’s RNA into DNA in a process called reverse transcription. This is because DNA is a lot more stable than RNA. The PCR machine then makes millions of copies of the DNA by running multiple “cycles” (like a washing machine). This process is called amplification and is extremely important in finding even the smallest amounts of DNA. As more cycles are run, more copies of the DNA are made —doubling every time it is copied —and making it easier to find. If the piece of DNA cannot be copied, there is no virus in the sample, or there is such a low amount that even this very sensitive test cannot detect it.

How do you know when a COVID-19 test is positive?

PCR tests tell you if the virus is detected (positive) or not (negative). Each PCR test has cutoff points (the number of cycles it runs), which tells the machine to stop running the test. It is important to note that different brands who make the PCR tests may have different cutoff values based on how sensitive the test is and how the test is designed. Additionally, laboratories across the province involved in COVID-19 testing use different testing kits.

At PHO, we have developed a PCR test in our lab, with positive and negative cutoff points. The cutoff point for a positive result for PHO’s developed lab test is 38 cycles. This means that if the virus is found at or before 38 cycles are completed, then the test is considered positive. The cutoff point for a negative result is 40 cycles. If the virus is detected between 38 and 40 cycles, we call this an indeterminate or inconclusive result. All inconclusive results are considered probable (likely) cases for public health reporting.  

What are cycle threshold values? 

The cycle threshold (Ct) value is the actual number of cycles it takes for the PCR test to detect the virus. It indicates an estimate of how much virus was likely in the sample to start with – not the actual amount. If the virus is found in a low number of cycles (Ct value under 30), it means that the virus was easier to find in sample and that the sample started out with a large amount of the virus. Think about it like the zoom button on your computer, if you only have to zoom in a little (zoom at 110%), it means that item was big to start with. If you have to zoom a lot (zoom at 180%), it means that the item was small to start with.

"Most samples that test positive at PHO are found after a low number of cycles are run. However, any indication of the virus in a sample is important, regardless of how many cycles it took to find."

— Samir Patel, Clinical Microbiologist and Deputy Chief Microbiology, Public Health Ontario

Can Ct values tell how serious an infection is?

Ct values are influenced by a number of factors including the PCR test kit, when the sample was collected, the machine used for testing, the technique of the health professional obtaining the sample and the type of sample (sampling method). In fact, different samples from the same person may result in different Ct values. Therefore, Ct values cannot actually tell you how severe of an infection you have or if you are more likely to develop severe disease – they simply indicate approximately how much virus was likely in the sample to start with. Several research studies are currently being done to look at the link between Ct values and severity of disease and infectiousness.

Although Ct values do not indicate the severity of disease, they may be able to provide important information for clinical and public health decision making when carefully analyzed with other factors such as type of test used, history of exposure to COVID-19, symptoms and individual characteristics of the patients. This analysis is completed by health care professionals and their testing laboratory, who have a deep understating of all factors being looked at.

Why aren’t cycle threshold reported on test results?

Like with other PCR tests (including non-COVID-19 tests), it is not recommended to provide Ct values on test results in Ontario (and Canada). PCR tests tell us if the virus is present or not in the sample provided to the lab; however, there are other factors to consider in interpreting lab results. Ct values are not directly comparable from one PCR test kit to the next, and can change with increased transportation times, sample storage conditions, and sample collection method. 

Because of this, Ct values can help support lab specialists in validating results as well as reviewing complex cases. However, they need to be considered alongside the other important factors we discussed earlier – like exposure history and individual characteristics. At PHO, Ct values are available to health care professionals upon request, and low level detected results (Ct value 35 to 38) are indicated on the laboratory report (since November 2020). We also have specialists who are available to health care professionals who have any questions on interpreting lab results or want to discuss complex cases. There is still a lot to learn about Ct values and more research is required to fully understand Ct values and their link to disease onset, severity and infectiousness.

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Updated 17 Feb 2021