That PCR is the most reliable method of identifying the coronavirus is something that has been repeated in the media lately. But do we know what it is? In this space, we explain it to you.
In the past few days, detection kits for COVID-19 have been on the media. After the controversy generated by the government purchase of more than 50,000 low-reliability tests and their consequent losses, reflection on understanding the detection methods of infected patients has become mandatory. What is PCR?
We will all have heard it: “the PCR method is the most complete and reliable”. Their high hit rate and minimal false positives have prompted the World Health Organization (WHO) to advise them as the main detection method.
Various spaces have covered the detection methods of this virus, but this time we want to take the opportunity to show you in detail what a PCR is and how it works. Keep reading and find out more about it!
👉 Sequencing the enemy
Coronavirus (COVID-19) is a viral agent that contains a single strand of RNA. In turn, it is classified as a positive single-stranded RNA. DNA and RNA are the most reliable ‘fingerprints’ that an organism can show.
The order of the nucleotides that form it reveals the identity of the individual, and there are many common regions for species and organisms. Consisting of a single information chain, the presence of coronavirus RNA in the body is unequivocal: if it is present in the patient’s sample, it is that it is infected.
Therefore, it has been of vital importance to sequence the genome of this virus since its discovery. Luckily, the first sample was genotyped on 11 January and you can see yourself through the page of the National Center for Biotechnology Information.
That amalgam of letters that you will be able to observe, corresponds to the order of the nucleotides of the RNA chain of the virus. Each nucleotide contains a nitrogenous base that corresponds to the represented letter:
- Guanine (g)
- Cytosine (c)
- In the case of RNA, uracil (u)
- In the case of DNA, thymine (t)
It will strike you then not to see a single one (u) in the coronavirus genome, right? Continue with us in this explanation, because it has an answer.
👉 PCR detects the intruder
Once the virus is sequenced, the effectiveness of the PCR comes into play. This technique, which dates back to the 80s and corresponds to the name Polymerase Chain Reaction, aims to amplify the DNA of a sample.
Yes, this is where the first virus trap lies: the coronavirus has no DNA if not RNA, which is why an even more sophisticated technique is required: RT-PCR, to transform the virus’s RNA into DNA.
To carry out this reaction, an enzyme called reverse transcriptase is essential. The process is the following:
From the patient’s sample, the enzyme reverse transcriptase can “identify” the RNA of the virus.
With nucleotides provided in the reaction mixture, the transcriptase enzyme will be able to generate a DNA strand complementary to that of the virus’s RNA. We have to see this enzyme as a worker: with the map of the virus’s RNA and the available nucleotides, it generates a new chain, in this case, DNA.
Here the polymerase enzyme comes into play and a normal PCR occurs. In short, the enzyme polymerase is another worker, which with available nucleotides is capable of generating thousands of copies of the transformed DNA strand.
This amplified DNA may be subjected to different techniques to determine whether it corresponds to the coronavirus genotype or not.
👉 Revealing identity
Once DNA is amplified, there are multiple techniques to assign it to a virus or organism. One of the simplest is agarose gel electrophoresis. We will use this example for simplicity, but there are sophisticated sequencers that do this.
DNA fragments have a negative electrical charge. Thanks to this, and applying an electric current in an agarose gel box, the different fragments will move along the gel during the time attracted by the positive pole. We have to see this as a race: the lightest DNA fragments arrive earlier, and the largest fragments stay halfway.
This is the key to detection: bands are formed at different distances on the gel. Taking a hypothetical example, if a mother and a child had different exact DNA fragments that were the same, the agarose gels of the two should present the same pattern, confirming their genetic relationship.
It is clear that the coronavirus detection methods are more sophisticated than the example shown, but we hope that with this explanation the operation of a PCR and its essential role in the detection of diseases will be clear.