How is Covid-19 tested? An overview of RT-PCR and rapid antigen tests

 

If you’re following the news on COVID-19 testing, you may have heard the debate about effective testing methods. The primary questions asked relate to the speed and accuracy of antigen testing versus RT-PCR testing. But what exactly are these methods? And what do the letters RT, PCR, qPCR and C_t stand for? How do they exactly operate?

 To answer these questions, it is first essential to understand a bit about DNA and RNA. DNA (or RNA in some cases) constitutes the genetic material of all living organisms. DNA and RNA belong to a class of molecules called as nucleic acids. These molecules contain hereditary information. This information is stored in the form of a code made up of four chemical letters – A, C, G and T or U. Each of these chemical letters constitute a nucleobase. These nucleobases pair with each other in a specific manner – A:T/U and C:G. Thus, combinations of A:T/U and G:C are called base pairs (figure 1). Nucleobases in the DNA are also attached to a phosphate molecule and a sugar molecule that serves as the backbone of the DNA. Together, the sugar, the phosphate and the nucleobase constitute what is known as a nucleotide. Nucleotides are arranged as two long helices (spirals) that gives DNA its characteristic “double helix” structure. DNA uses the letters A,C,G,T and RNA uses A,C,G,U. They are long ladder-like molecules that have a different nucleobase at each rung in the ladder (figure 2). 

                          

                                                  Figure 1: The four nucleobases

For easy comparison, DNA can be compared with a spiral staircase or a ladder. RNA looks like only half of the staircase. DNA is double-stranded and RNA is single-stranded. 

      

                                        Figure 2: The double helical structure of DNA

 Back to the virus. A virus has either DNA or RNA as its genetic material. The coronavirus uses RNA. PCR-based tests target the genetic material, i.e. the RNA for coronavirus. Successful detection of viral RNA molecules in a sample means that a test is positive.  

 How does this work? Let’s start with the “RT” part in RT-PCR. Because RNA is a “half-ladder,” the first step that’s needed is to turn it into a full double-stranded ladder. This is done by a process called reverse transcription or RT (figure 3). This reaction uses an enzyme (blue) to copy the RNA code into a single strand of DNA. The enzyme that does this needs a running start, and so the reaction uses a “primer” (in green) that provides this starting point. 

                                                 

                                                       Figure 3: The RT-PCR process

Once the RT step has converted RNA into DNA, we’re ready for the PCR step. PCR stands for polymerase chain reaction. This works like RT, but now DNA is copied into more DNA. Again, primers bind to the DNA and a polymerase makes a copy. 

What makes this useful for clinical diagnostics is in the design of the primers. These are made so that they recognize a part of the virus genome that is unique to that virus. So, the primers should only bind and start the PCR reaction if the right code is present.

 The other thing that is powerful is that this reaction is exponential. One molecule of DNA is copied into 2, then 4, then 8, then 16, then 32…up to millions or more. Each time this doubling happens is the result of a “cycle” of PCR (figure 4). 

 

                                                Figure 4: Each DNA molecule is replicated exponentially

 This is where the “q” becomes important – it stands for quantitative. This means that as the PCR reaction goes on, a machine is monitoring, or quantitating, the amount of DNA that is being made. This is usually done using a dye that binds to the double-stranded DNA and lights up, emitting fluorescence.   

Because the reaction is exponential, the amount of DNA made with each cycle looks like the green line in this graph – it starts out by doubling with each cycle, and then tapers off as the reaction runs out of primers to make new strands. The Ct value is the number of cycles required to reach a threshold of detection (figure 5). Why this matters is that the more RNA or DNA that there is in the sample, the fewer cycles are needed to make enough to be detected. So, the lower the Ct value, the more viral RNA was present. 

                            

      Figure 5: The fluorescence emitted in the PCR is dependent on the quantity of DNA/RNA amplified 

 One of the challenges with this testing arises because PCR is just really efficient. Even a small amount of contamination can be amplified after lots of cycles. Or, if the primers bind to stray DNA or RNA besides viral genome, that can also start making DNA and give a signal.

 Thus, one of the important metrics in RT-qPCR tests is the Ct cutoff. This is how low the Ct value needs to be for a sample to be considered “positive.” Every sample that is tested technically gives a Ct number, but then testing centers interpret this as positive or negative.

The lower the cutoff for a test to be called positive, the more likely a test is to miss people who are actually infected. But, the higher the cutoff, the more likely a test is to give false positive results or flag people who are well on their way to recovery.When it comes to setting Ct cutoffs, there are no easy answers, especially as scientists are still learning about how viral load varies over the course of an infection and how this shows up in different types of samples like nasal swabs or saliva.

Now what is the antigen test? Antigen tests detect the protein molecules that encapsulate the DNA/RNA of the virus. To do this, there are specific molecules called antibodies that bind to the protein “antigens” of the virus. Binding of antibodies to the antigens is usually accompanied by a colour change for easy detection. Antigen tests are rapid (around 30 mins) to perform and are point-of-care tests. However, a big drawback is that it is not always possible to detect the viral proteins in this test. Antigens tests are rapid but can give false negatives; RT-PCR tests are time consuming but are more accurate. Hence, it was proposed that all people testing negative in the antigen tests should undergo a RT-PCR test.

 This post was a bit long, but, the next time you hear about RT-PCR testing and Ct values, you’ll be ready to talk about what all of these letters mean and the role they play in trying to control the COVID-19 pandemic.