How is RT-PCR used to test for COVID-19?

7th Jan 2021

Quick and accurate diagnosis of COVID-19 is important. Let’s look more closely at the gold standard for diagnosing COVID-19—real-time reverse transcription polymerase chain reaction, or RT-PCR. 

 

What is RT-PCR?

RT-PCR is a laboratory-based technique used for detecting and comparing the levels of ribonucleic acid (RNA) and the surface proteins in a sample, particularly samples with limited quantities of RNA. 

 

How can we use RT-PCR to diagnose COVID-19

Obtaining a sample

Diagnosing COVID-19 requires taking a sample from the patient, usually using a nasopharyngeal or oropharyngeal swab, or more recently a saliva sample, and testing for the presence of viral RNA using RT-PCR.

Man getting nasopharyngeal swab. SARS-CoV-2 virus. Illustration.

Figure 1. Real-time reverse transcription polymerase chain reaction (RT-PCR) can test for the presence of SARS-CoV-2 RNA in a sample from a nasopharyngeal or oropharyngeal swab or a saliva sample. 

Isolating RNA from the sample and creating complementary-DNA (cDNA)

Because SARS-CoV-2 contains only RNA, the first step is to extract and isolate all the RNA from the patient’s sample. But RNA is more difficult to work with in the lab than DNA. And DNA can be amplified and quantified using a relatively simple, accurate method called the polymerase chain reaction, or PCR. But PCR doesn’t work with RNA! 

So, we convert the RNA to DNA using an enzyme called reverse transcriptase. The resulting DNA is known as complementary-DNA, or cDNA, because its sequence is complementary to that of the original RNA strand. 

RNA strand. Complementary DNA strand. Illustration.

Figure 2. In the first step of real-time reverse transcription polymerase chain reaction (RT-PCR), RNA in a sample is converted to complementary-DNA (cDNA) using reverse transcriptase. 

Isolating SARS-CoV-2 cDNA

Now, it’s important to know that at this stage we’ve actually created a collection of cDNAs that represent all the RNA that was present in the original sample. This could include RNA from bacteria or other viruses, or from the patient’s own cells. So, we need a way to determine whether SARS-CoV-2 cDNA is present in the sample. 

For that, we use polymerase chain reaction (PCR), which allows us to amplify and detect a specific DNA molecule—the viral cDNA. 

PCR involves 3 basic steps, which are repeated up to 40 times:

  1. Denaturation
  2. Annealing
  3. Elongation

 

Denaturation

In the first step, all the double-stranded molecules are denatured, meaning the two strands are separated. 

Double stranded molecule. Separated DNA molecule into single strands. Illustration.

Figure 3. The first step of the polymerase chain reaction (PCR) process occurs once RNA has been converted to complementary-DNA (cDNA) by reverse transcriptase. Then the two strands are denatured, or separated. 

Annealing

In the second step, a pair of primers that are specific to the SARS-CoV-2 cDNA anneal, or attach, to each cDNA strand. The primers are short nucleotide sequences that are complementary to a unique sequence in the viral cDNA. The specificity of the primers ensures that they only bind to the viral cDNA and not to any of the other cDNAs present in the sample. 

Viral cDNA with primers bound. Other cDNA with no binding of primers. Illustration.

Figure 4. The second step of the polymerase chain reaction (PCR) process is called annealing. It occurs once the complementary-DNA (cDNA) and RNA strands are denatured. Short nucleotide sequences called primers attach to the viral cDNA. 

Elongation

In the third step, or elongation, an enzyme known as a polymerase adds nucleotides to the ends of the primers, using the original DNA strand as a template, to create two double-stranded DNA molecules! 

Two single stranded complementary DNA strands. Two double stranded complementary DNA strands. Illustration.

Figure 5. During the elongation phase of polymerase chain reaction (PCR), each viral complementary-DNA (cDNA) strand and primer are acted on by the enzyme polymerase to add nucleotides and build double-stranded cDNA molecules. 

These three steps—denaturation, annealing, and elongation—are then repeated, with the amount of DNA doubling in every cycle. So, if we started with only one cDNA molecule, after 35 cycles we would have 235 or over 34 billion identical DNA molecules! 

Figure 6. After each time the three steps of polymerase chain reaction (PCR)—denaturation, annealing and elongation¬—are performed, the amount of DNA in a sample doubles.

Figure 6. After each time the three steps of polymerase chain reaction (PCR)—denaturation, annealing, and elongation­—are performed, the amount of DNA in a sample doubles. 

 

Clickable call to action, "Start learning for free", with direct link to sign up for a free Medmastery trial account.

 

What is real-time RT-PCR?

In real-time RT-PCR, a probe is added during the PCR process, which gives off fluorescence whenever a new DNA molecule is formed. 

DNA strands with fluorescent tag. Illustration.

Figure 7. In real-time reverse transcription polymerase chain reaction​ (RT-PCR), a probe is added during the PCR process, which will fluoresce whenever a new DNA molecule is formed. 

The increase in viral cDNA can be followed in real-time by tracking the increase in fluorescent signal. When the level of fluorescence exceeds a certain threshold, we can be confident that the signal is significantly above the background level. 

The cycle threshold (Ct) value is the number of cycles required for the fluorescent signal to exceed that specific threshold value. The lower the Ct value, the more RNA was present in the original sample, indicating a higher viral load. For SARS-CoV-2, a Ct value of less than 40 is considered a positive result. 

Graph of fluorescence vs number of PCR cycles. Illustration.

Figure 8. When using real-time reverse transcription polymerase chain reaction​ (RT-PCR), the number of cycles it takes for fluorescence to exceed a specific threshold value above the background level is the cycle threshold, or Ct, value. Lower Ct values indicate a higher viral load in the original sample. 

If the result is positive, it is also important that the Cvalue be reported, since this indicates the patient’s viral load or infectiousness. Several studies have shown that viral load—measured by Ct value—can help predict disease progression.  

For example, a study of hospitalized patients showed that those who developed more severe disease had a lower Ct value on their RT-PCR test done at admission than those who developed more mild disease.1 They also showed that the probability of disease progression correlated with the Ct value on admission.1

Therefore, as a clinician, you would not only want to know if your patient was positive on RT-PCR but also by how much. What was their cycle threshold value? Because a patient with a Ct value of 10 has one million times as many viral particles in their throat as compared to a patient with a Cvalue of 30.

Two men with COVID-19. Illustration.

Figure 9. A COVID-19 positive patient with a cycle threshold (Ct) of 10 has a higher viral load than a COVID-19 positive patient with a Ct of 30. In fact, the first patient will have one million times as many viral particles as the second patient. 

 

How long does it take to get the results of RT-PCR testing?

Obtaining quick, accurate test results is important to prevent transmission of the virus.

So how long does RT-PCR take?

A typical real-time RT-PCR run takes approximately 2–4 hours, including the time it takes to extract and reverse transcribe the RNA. The specific equipment available and the level of automation of the process is key to determining how quickly a specific lab can produce results.

Because time is of the essence during a pandemic, many companies have developed newer RT-PCR kits that can produce results in as little as 15 minutes! Although many of these still need to be validated.

How accurate is RT-PCR for detecting SARS-CoV-2?

Obtaining quick results is great, but it’s just as important that these results can accurately identify people with the disease, so they can be isolated to reduce transmission. So, what is the accuracy of RT-PCR testing? 

To answer that question, we need to consider both the analytical specificity and sensitivity—the ability of the RT-PCR assay itself to detect the viral RNA when it’s present in the sample—as well as the clinical sensitivity and specificity —the ability of the test to detect individuals who have or don’t have the disease. 

It’s the clinical specificity and sensitivity that ultimately determines the rate of false positives—individuals who test positive but aren’t actually infected—and false negatives—individuals who test negative but are actually infected with the virus. 

RNA. Test tube. Man. SARS-CoV-2 virus. Illustration.

Figure 10. To determine how accurate RT-PCR is in detecting COVID-19, consider a) the analytical specificity and sensitivity, or the ability of a test to detect viral RNA in a sample, and b) the clinical specificity and sensitivity, or the ability of the test to detect individuals who have or don’t have the disease. 

The good news is that RT-PCR has a high analytical and clinical specificity for SARS-CoV-2, meaning that it produces few false-positive results. So, when a patient tests positive for the virus, we can be fairly certain they are actually infected.  

When they do occur, false positives are generally the result of technical errors or reagent contamination, and are generally avoidable with good laboratory technique and the use of proper testing controls.

Man with COVID-19. Illustration.

Figure 11. RT-PCR has a high analytical and clinical specificity for SARS-CoV-2, meaning the false positive rate with this test is low. 

The analytical sensitivity of SARS-CoV-2 RT-PCR assays is still up for debate, but it’s generally thought to be high.

However, the clinical sensitivity of RT-PCR for SARS-CoV-2 is only around 70-80% at best (one study estimated it to be as low as 38%). This means a single negative test result doesn’t always mean the patient doesn’t have the virus.

Warning sign. Man with COVID-19.

Figure 12. The clinical sensitivity of RT-PCR for SARS-CoV-2 is only 70-80% at best. So a single negative test does not always mean the patient doesn’t have the virus. 

False negatives are a big problem during a pandemic, since these patients actually have the virus, but they think they’re in the clear, so they may engage in behaviors that cause them to transmit the virus to others.

But what could be causing false negatives in COVID-19 testing?

Although most RT-PCR kits use primers that target regions of the viral RNA that don’t appear to change much, variations or mutations in the viral RNA sequence could lead to false-negative results by preventing proper primer binding. 

And not all SARS-CoV-2 RT-PCR test kits are created equal, with some being more sensitive than others. 

But ultimately, any RT-PCR assay is only as good as the sample put into it. 

So, the sample collection process is probably the biggest contributor to false-negative results. This could include improper sampling technique, transport, or storage, but the type of specimen used and the timing of sample collection most likely have the greatest influence on the overall sensitivity of SARS-CoV-2 RT-PCR tests. 

Primer and DNA strand. Test tube. Hand holding swab. Grocery cart. Refrigerator. Swab. Sputum. Lungs with bronchoscope. Calendar. Illustration.

Figure 13. The causes of false negatives in reverse transcription polymerase chain reaction​ (RT-PCR) testing.

 

That’s it for now. If you want to improve your understanding of key concepts in medicine, and improve your clinical skills, make sure to register for a free trial account, which will give you access to free videos and downloads. We’ll help you make the right decisions for yourself and your patients.

References

  1. Yu, X, Sun, S, Shi, Y, et al. 2020. SARS-CoV-2 viral load in sputum correlates with risk of COVID-19 progression. Critical Care24: 170. PMID: 32326952

Recommended reading

  • Bustin, SA and Nolan, T. 2020. RT-qPCR testing of SARS-CoV-2: a primer. Int J Mol Sci21: 3004. PMID: 32344568
  • ​Corman, VM, Landt, O, Kaiser, M, et al. 2020. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill25: 2000045. PMID: 31992387
  • Tahamtan, A and Ardebili, A. 2020. Real-time RT-PCR in COVID-19 detection: issues affecting the results. Expert Rev Mol Diagn. 5: 453–454. PMID: 32297805
  • Zhang, J, Gharizadeh, B, Lu, D, et al. 2020. Navigating the pandemic response life cycle: molecular diagnostics and immunoassays in the context of COVID-19 management. IEEE Rev Biomed EngPMID: 32356761