Are we testing viruses in the wrong way?
By: Dr. Alessandro Crimi
Tags: Future Technology, Biology, Covid 19, Medicine, Health

1. Polymerase Chain Reaction (PCR)

PCR is probably the most commonly used method for genetics diagnostic. It rapidly makes several of copies of a specific DNA sample, to be subsequently studied. This generally means to “see a bar on a gel”, and therefore conclude the presence of a specific DNA. Briefly, if a DNA strain is present in a sample (e.g. related to a virus or known gene), it is amplified, and then seen on a gel. If it is not present the test is negative (no amplification and nothing significant on the gel). The diagnostic purpose is not the only one as the technique is also used to DNA and gene cloning, gene mutagenesis, construction of DNA-based phylogenies, etc. If you are a biologist/pathologist, you can probably skip this section. If you are new to this, prepare yourself to have a little headache, and this is

why I do believe in the future we will NOT use this technique.

For a general reader the required steps might appear cumbersome, and indeed they are. The third section describes a simpler protocol. PCR was invented in 1984 by the American biochemist Kary Mullis. A peculiar character — surfer and fan of LSD — who won the Nobel Prize in the 1993 for this technique [1]. Since then, it has been useful in infinite contexts. Several improvements and evolutions of this technique exist, as digital PCR (dPCR), quantitative PCR (qPCR), and asymmetric PCR. The basic idea of PCR is to perform 3 steps cyclically [2]. Those are changes of temperatures hence the method is also called thermal cycling:

(1) denaturation of the DNA template into single strands;

(2) annealing of primers to each original strand for new strand synthesis; and

(3) extension of the new DNA strands from the primers.

In the denaturation step, two strands of the DNA double helix are physically split (generally at 94–98 °C). In this way, by breaking the hydrogen bonds between complementary bases 2 single strand DNA are obtained. The annealing step while lowering the temperature to 50–65 °C allows primers (single strands of initial match) to bind to the complementary sequences of DNA. This opens to the replication of strands of DNA (elongation) by adding free dNTPs (Nucleoside triphosphate) and creating new templates for the polymerase at 72–80 °C. Polymerase is the enzyme involved in the process). Therefore, given several cycles (e.g. 30) the original DNA template is exponentially amplified. In this process four main reagents are necessary: the DNA sample, the primers (which are 2 short single strand DNA fragments known as oligonucleotides complementary sequence to the target DNA region), nDTPs and a DNA polymerase (and a machine to change quickly and stably the temperatures). Luckily, nowadays you can use mastermix which already include most elements. The most used DNA polymerase enzyme is the Taq polymerase which is heat-stable and originally isolated by the thermophilic bacterium Thermus aquaticus. If the enzyme was not heat-stable, it should be added at every cycle. Primers are 2 since 1 per side is necessary (as shown below), and they are specific to what we are testing (there are primers for malaria, primers for COVID, primers for HIV, etc). Once we know the specific primer for what we want to test we can order it via specific online tools as IDT-primer quest.

The settings can be summarized in the following picture, representing on the left the ingredients and on the right the aforementioned cycles.

Credits: AgaKahn Academy

However, after the reaction, the “cocktail” still needs to be inserted in an agarose solution with TAE buffer contained into an electrophoresis chamber to allow visualization:

This has also been the most used way to detect COVID-19. A sample from a patient is collected (e.g. from a swab inserted in the nose or throat) and then analyzed by doing a PCR. To further complicate the situation, we have to bear in mind that viruses have only RNA, so first we have to create a DNA by complementing the possible RNA. Moreover, with a variation of PCR called RT-PCR, it is possible to quantify the viral load. All those steps are also summarized in the following video:

As you can imagine, given so many steps and conditions (primers sequence, temperature, etc.), many things can go wrong, and this is why often many people in the last 10 months reported negative results to this test, though they probably had the COVID19. Also, some people were told at some point to be cured, but instead they were not, and therefore if they were afterwards again sick, they believed having contracted the virus again. The estimated success rate was about 60% … yes 1 over 3 times it might not work. The main reason for this low sensitivity is believed to be related to the viral load collected by the swabs. Namely, that it is difficult to collect at the right moment viruses on the throat or nasal cavity by using swabs [3]. Also, the required time is about 2 hours, though in clinical settings, results to patients were generally given within 24–48 hours. 2 hours might seem little, but in large scale situations as those we faced, it is a huge amount of time.