If you’re following the news on COVID-19 testing, you may have heard the debate about RT-PCR testing and Ct values leading to false positive results. You may be wondering “How does PCR work, what makes it RT, and what the heck is a Ct value?” A #SciCommSunday primer.
I’m actually going to start by making things a bit more complicated, but stay with me – this will help in the long run. The “RT-PCR” test is really an RT-qPCR test, and the “q” is what is at the center of the debate.
So, what do all of those letters mean? To answer that, we have to look at a different set of letters. Every virus has what is called a genome – this is the code that carries all of the instructions it needs to infect a cell and get that cell to make more copies of the virus.
The code is made up of four letters – A, C, G, and T or U. These are each a different “nucleobase” with a unique chemical structure. These nucleobases can pair with each other, and they usually do so in a pattern of C:G and T/U:A. This allows the code to be copied and read. 
There are also two different types of molecules that can carry this code, DNA (which uses A,C,G,T) and RNA (which uses A,C,G,U). They are long ladder-like molecules that have a different nucleobase at each rung in the ladder.
You probably noticed that while DNA looks like a ladder, RNA looks like only half of a ladder. This will be important later. We say that DNA is double-stranded and RNA is single-stranded. There are other chemical differences, but we don’t need to worry about those here.
Because each virus has a unique code in its DNA or RNA genome, the PCR-based tests are designed to look for that specific code and only produce a signal if that code is present. But, of course, that process is not perfect.
How does this work? Let’s start with the “RT” part. Viruses can use either DNA or RNA for their genome. The SARS-CoV-2 virus that causes COVID-19 happens to use RNA. Because RNA is a “half-ladder,” the first step that’s needed is to turn it into a whole double-stranded ladder.
This is done by a process called reverse transcription or RT. 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.
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.
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.
Because the reaction is exponential, the amount of DNA made with each cycle looks like the blue 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. 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.
One of the challenges with this testing arises because PCR is just really darned efficient. Even a small amount of contamination can be amplified after lots of cycles. Or, if the primers bind to something 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.
But, now 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.
