RT-QPCR TESTS

RT-qPCR tests (or PCR tests for short), rely on the presence of the Coronavirus RNA (their version of DNA). The process takes a fluid sample from the patient, turns it into DNA, and attempts to duplicate this DNA times over. After the process, if the virus RNA was present there will be a high quantity of new DNA that is visible, showing that the sample contained Coronavirus.

“PCR Machine, Replication, Microcentrifuge Tube.” Straits Times, www.straitstimes.com/singapore/from-polymerase-chain-reaction-to-antibody-tests-a-look-at-covid-19-tests-and-their-uses.

TESTING PROCESS

The first part of RT-qPCR is the RT: Reverse transcriptase. This is an enzyme that helps take the RNA of the virus, and makes copies of it in DNA form. We need to do this as one enzyme in the PCR process, DNA polymerase, only recognizes DNA, meaning no replication would occur if we tried to work straight from virus RNA.

Once this is done, the PCR process can begin. There are a couple of important things to remember about DNA for this process. First, all DNA is made up of molecules called nucleotides, that generate a sequence. Each of these molecules is linked together to form a long strand of DNA. A paperclip chain would be a DNA strand, while a single paperclip would be a nucleotide.

Second, DNA sequences are unique. The DNA we create from the virus's RNA has a sequence that is not found in human DNA. We can take advantage of this by creating primers that only recognize the viral DNA sequence. Primers are just small pieces of DNA, and they are where we start to build a new, long DNA strand. Think of them as short paperclip chains. Once these primers have stuck onto the viral DNA, it allows the viral DNA and only the viral DNA to replicate, while any human DNA in the sample is unaffected.

Third, DNA is double-stranded. Each of these paperclip chains has another chain that it is stuck to, and we can imagine it as if the paperclips were magnetized. These individual paperclip chains (DNA strands) are much stronger than the attraction between the two chains. Because of this, we are able to separate the two into two single chains, while each individual chain remains intact.

Check out this video of the PCR process. After the video, you can read about each step below.

Doctorbhanuprakash, director. Polymerase Chain Reaction Animation. YouTube, YouTube, 29 Oct. 2019, www.youtube.com/watch?v=ClhUe53W7xw.

Let's start our PCR process. To our DNA we are going to add individual nucleotides (paperclips! a.k.a. dNTPs), primers, and DNA polymerase. We'll get to these in a moment.

1. The first step is to heat up the DNA, which pulls the two strands apart. This process is called Denaturation.

2. The second step is to cool things down, just a little bit. This allows our primers to stick onto the long DNA strands. This step is called Annealing.

3. Once the primers are stuck, DNA polymerase comes into play. DNA polymerase is an enzyme that builds our new paperclip chain. It takes the nucleotides, and starts linking them to the end of our paperclip chain. It will keep going until it reaches the end of the strand. This step is called Extention.

Once this is done, you've now turned a single DNA strand back into a double strand! But don't forget, you started with two single strands. This process was happening at the same time to both strands, meaning that you've now doubled the DNA you began with. Every time you repeat this cycle, you double the amount of DNA present, as the process happens over again to each new DNA strand. If you started with one piece of DNA, by the time you completed twenty cycles of this process, you'd have over 1 million!

This is a standard PCR process. The only difference between this process and the qPCR process used in COVID testing is that a dye is added to the mix. This dye is only visible when stuck onto DNA molecules, so when enough DNA replication has occurred and lots of DNA is present, it allows us to visually see that there is a large amount of DNA. Keep in mind that this process will only work in COVID testing if the virus is present. Without the virus, we will have nothing to replicate, so no matter how many times the cycle goes, it will show no result. This would be a negative test result, showing that no virus was present in the sample, meaning the patient was not infected.

“PCR DNA Replication Process.” Wikipedia, en.wikipedia.org/wiki/Polymerase_chain_reaction.