Polymerase Chain Reaction: how molecular tests work

With the advent of the 2020 pandemic, we have often heard of the molecular PCR technique, an acronym for DNA CHAIN ​​REACTION POLYMERASE, so much so that it has become an extremely inflated acronym in common language. PCR technology has revolutionized the activity of research and diagnostic laboratories by finding applications and uses in various fields of medicine and biology. But what is PCR? How does it work? Who was its creator?

Kary B. Mullis Nobel Prize in Chemistry in 1993

Let’s start with the last question: PCR was conceived in 1983 by Kary B. Mullis who was awarded the Nobel Prize for Chemistry (1993) for this. Mullis was an American scientist, a biochemist to be exact, born in 1944, a man born of his times, a man who lived between genius and recklessness, a scientist with deep curiosity, eccentric, and perhaps the only Nobel laureate who hypothesizes an encounter with aliens. ( Dancing naked in the field of the mind, a sparkling volume that talks about various themes, from the scientific method to parapsychology, to HIV and AIDS, to the case of OJ Simpson).

It was the spring of 1983 and as he himself later confessed: «Sometimes good ideas come precisely when you are not looking for them. […] I could hardly sleep that night, with the “deoxyribonucleic” bombs exploding in my brain. ” He died on 7 August 2019 at the age of 74 and we cannot help but wonder what his position would have been in relation to Covid-19.

What is PCR

Although the technique has seen improvements over the years, the intuition and therefore the basic principle has remained the same: rapidly obtaining millions of identical DNA molecules from extremely small quantities of nucleic acid, in other words, an amplification reaction. in vitro of a specific DNA fragment by means of an enzyme: DNA polymerase.

It is not the purpose of this article to illustrate the biochemical mechanisms of the molecular technique, however the following figure, albeit extremely simplified, can be useful at a conceptual level also for “laymen” and serves us to answer the second question ” how Works? “.

The number of new DNA molecules increases with each cycle. In fact, during the first cycle, two molecules are obtained from a single DNA molecule, each consisting of an “old” helix which was the template for the synthesis of the “new” helix; at the second cycle each of the two molecules is denatured, the four DNA strands obtained from the template for the activity of the DNA polymerase and in the end, the DNA molecules become four. The amplification process proceeds in this way from cycle to cycle, so assuming 36 cycles … we would get 68 billion copies !!!!

At this point we have understood what PCR does, let’s now adapt this concept to the molecular buffer. We have described how this type of swab works in a previous article (link): it highlights the genetic material of the virus, its unique and unmistakable “signature”.

But the Molecular test with PCR, is it diagnostic or not? As a reflection proposal, we report below a study, published on the JAMA Network, a viewpoint Interpreting Diagnostic Tests for SARS-CoV-2, which describes how to interpret the results and variations overtime of 2 types of diagnostic tests commonly used for the diagnosis of SARS-CoV-2 infection:

• the reverse transcription reaction of polymerase chain (RT-PCR)
• IgM and IgG with enzyme-linked immunosorbent assays (ELISA)

Time-Estimated Change in Diagnostic Tests for Detecting SARS-CoV-2 Infection in Relation to Onset of Symptoms

The study shows that in the majority of subjects with symptomatic COVID-19 infection, viral RNA, collected by nasopharyngeal swab, becomes detectable from the first day in which symptoms are present, with a peak within the first week from ‘ onset of symptoms. This positivity in mild cases begins to decrease by the third week and subsequently becomes undetectable. Conversely, in seriously ill hospitalized patients, CRP positivity may persist beyond 3 weeks after disease onset. However, a “positive” PCR result reflects only the detection of viral RNA and does not necessarily indicate the presence of viable viruses.

Having the molecular PCR test a specificity equal to 100% it remains stable whether a molecular buffer Rt-PCR is able to give us a diagnosis confirmation from Covid-19 or not.