Testing for COVID-19 using RT-PCR

In this video, you'll take a deep dive into the RT-PCR process for SARS-CoV-2. You'll learn how the process works and how it can go wrong. You'll also learn to watch for a significant value that may help you predict disease progress.

Franz Wiesbauer, MD MPH
Franz Wiesbauer, MD MPH
9th Jul 2020 • 6m read

COVID-19 is diagnosed using the real-time reverse transcription polymerase chain reaction (RT-PCR). In this video from our COVID Mini: Diagnostics course, you'll take a super deep dive into the RT-PCR process for SARS-CoV-2. You'll learn how it works and how it can go wrong. You'll also learn to watch for a significant value that may help you predict disease progress.

Join our COVID Mini: Diagnostics course now!

Clinicians and scientists are scrambling to produce evidence-based studies that will help to guide the identification, testing, and treatment of this horrible disease. In this course, we'll sift through the evidence to show you how to diagnose COVID-19 using laboratory tests and CT scans, and outline the benefits and limitations of each.

Start the first chapter of our COVID Mini: Diagnostics course for free

Video transcript

Diagnosing COVID-19 requires taking a sample from the patient, usually using a nasal pharyngeal or oral pharyngeal swab, or more recently, a saliva sample, and testing for the presence of viral RNA using real time reverse transcriptase polymerase chain reaction, or RT PCR. So how does RT PCR work? The first step is to extract and isolate all the RNA from the patient 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. Now, it's important to know that at this stage, we've actually created a collection of C, D, and A's 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 the SARS cov to see DNA is present in the sample. For that, we use PCR which allows us to amplify and detect a specific DNA molecule. In this case, the viral cDNA PCR involves three basic steps which are repeated upon 40 times.

In the first step, all the double stranded DNA molecules are denature ated, meaning the two strands are separated. In the second step, a pair of primers that are specific to the SARS cov, two cDNA and Neil or attached to each DNA 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 cDNA is present in the sample. In the third step called elongation, an enzyme known as a polymerase adds nucleotides to the ends of the primers using the original DNA strand as a template.

And now we've gone from one double stranded DNA molecule to to these three steps denaturation. annealing and elongation are then repeated with the amount of DNA doubling in every cycle. So if you started with only one cDNA molecule, after just 35 cycles, you would have two to the power of 35 or over 34 billion identical DNA molecules.

In real time PCR probe is added during the PCR process, which gives off fluorescence whenever a new DNA molecule is formed. The increase and viral cDNA can be followed in real time by tracking the increase in fluorescence signal. When the level of fluorescence exceeds a certain threshold, we can be confident that the signal is significantly above the background level.

The CT, or a cycle threshold value is the number of cycles required for the fluorescence 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 to a Ct value of less than 40 is considered a positive test.

If the result is positive, it is also important that the Ct value be reported, since this indicates the patient's viral load or infectiousness. Several studies have shown that viral load as measured by Ct value can help predict disease progress. For example, this study of hospitalized patients showed that those who develop more severe disease had lower Ct values on their initial RT PCR test on admission than those who develop more mild disease.

They also showed that The probability of disease progression correlated with the Ct value on admission. 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 initial cycle threshold value. Because as we have seen, a patient with the cycle threshold value of 10 has 1 million times as many viral particles in their throat, as compared to a patient with a cycle threshold value of 30.

Obtaining quick, accurate test results is important to prevent transmission of the virus. So how long does RT PCR take? A typical real time PCR run takes approximately two to four 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. 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's the accuracy of RT PCR testing? To answer that question, we need to consider both the analytical specificity and sensitivity that is 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 which tells us how well the test detects 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. The good news is that RT PCR has a high analytical and clinical specificity for SARS cov.

Two, meaning that it produces few false positive results. So when a patient tests positive for the virus, we can be fairly certain that 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.

The analytical sensitivity of SARS cov to 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 two is only around 70 to 80%. At best, One study estimated it to be as low as 38%. This means a single negative test results does not always mean the patient doesn't have the disease.

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 these false negatives. 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. To RT PCR test kits are created equal, with some being more sensitive than others. But ultimately, any RT PCR essay 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 to RT PCR tests.

We will examine this in more detail in the next lesson. So hope you liked this video. Absolutely, make sure to check out the course this video was taken from and to register for a free trial account which will give you access to select the chapters of the course. If you want to learn how Medmastery can help you become a great clinician, make sure to watch the about mastery video. So Well thanks for watching and I hope to see you again soon.