Corona test: which tests are available and how reliable are they?

Corona test: which tests are available and how reliable they are.

It’s a back and forth: Who will even get a corona test? What does PCR mean? And why not just test all of them? There’s a good reason for that.

How does the PCR test work?

PCR stands for the English term polymerase chain reaction, in German polymerase chain reaction. It was developed in 1983 by Kary Mullis. Mullis received the Nobel Prize in Chemistry for this in 1993, because the development was a milestone in molecular biology research. Still, he was a weirdo – he denied, among other things, that HIV is responsible for AIDS and man-made climate change as well.
The set-up for a PCR
The PCR is a system with which specific DNA sequences can be multiplied or copied outside the living organism, in vitro. To do this, enzymes and building blocks are used that are also responsible for doubling the DNA in the body’s cells. This happens in the body during cell division, for example during the development of the embryo.

The DNA that you want to replicate is often referred to as the starting DNA. At the beginning of the process, it is placed in a reaction vessel together with the replication enzymes and building blocks. This mix of molecules always contains very similar substances.

On the one hand, the individual “DNA letters” adenine, guanine, thymine and cytosine. The DNA is made up of these building blocks. In addition, there is a so-called DNA polymerase, an enzyme that can put these building blocks together. Then there are the primers. They show the polymerase where to start assembling the DNA building blocks.

The DNA is put into a reaction vessel together with the DNA letters, the polymerase and the primers. For example a small tube. This is then put into a so-called thermal cycler. This is a device that can automatically change the temperature and both heats and cools the tube during the PCR.

The process of the PCR
The basic principle of PCR is relatively simple and is based on the fact that the various steps of the polymerase chain reaction only take place at certain temperatures.

Denaturation
First of all, the starting DNA that you want to replicate is denatured. To do this, the reaction vessel is heated to 94 to 96 degrees Celsius in the thermal cycler.

This causes the two strands of DNA to separate from each other. DNA is a double helix made up of two complementary strands. Complementary means that the structure of the two strands is dependent on one another and one can always deduce the structure of the other from one of the two strands of DNA.

After denaturation, the DNA is therefore present as a single strand. It is now, so to speak, free to be re-paired.

Attachment
Now the temperature is reduced to below 72 degrees Celsius. The primers become active. The primers are also complementary to the DNA, but only in a very small section to which they can now attach.

If the primer cannot find an exactly matching stretch of DNA, it cannot attach. The primers are therefore gene-specific. In the case of the corona tests, they are matched to certain genes of the SARS-CoV-2 virus. Namely, on genes that only occur in this form in SARS-CoV-2.

renewal
After the attachment phase, the DNA is elongated at around 72 degrees Celsius. Starting from the primers, the polymerases attach a new strand to the exposed strands of the starting DNA. New double strands are formed. A starting double-stranded DNA becomes two.

This completes the first cycle of the PCR, consisting of denaturation, attachment and extension. In order to continue to replicate the DNA, the temperature is simply raised by the thermal cycler to 94 degrees Celsius and the process starts all over again. The amount of DNA grows more and more because a larger number of templates are available each time. Hence the term “chain reaction”.

There are slight variations in performing PCRs. The temperatures of the respective steps can vary slightly, for example, depending on the primers used. In cases, we also do accumulation and elongation in the same temperature step.

From viral RNA to DNA
However, the corona virus does not have DNA, but RNA. The genetic material is therefore in a different form. The corona test is therefore not a simple PCR, but an RT-PCR. RT stands for reverse transcriptase. An enzyme that can transcribe RNA into DNA. This happens in one step before the actual PCR, but in the same reaction vessel.

Just like polymerase, reverse transcriptase also needs a primer to help it find a starting point. Starting from the primer, the reverse transcriptase then attaches the complementary DNA building blocks to the virus RNA. The resulting DNA strand contains the same genetic information as the virus genome.

After the DNA-RNA double strand has been separated by heating, the DNA strand is used as a template for the PCR. After that, the cycles run like normal PCR.

Here, too, there are slight variations in the process, similar to normal PCR.

Luminous genes as a distinguishing feature
However, the corona test has another special feature. It is a so-called real-time PCR (it is abbreviated with a q or r. For the corona test, for example, RT-qPCR, sometimes also qRT-PCR). This means that you can see during the runtime whether there are SARS-CoV-2 genes in the sample. This works via fluorescence.

With real-time PCR, so-called DNA probes are located in the reaction vessel in addition to the gene-specific primers. Like the primer, these probes are gene-specific and only bind to the coronavirus gene segment sought during the deposition phase in which the primer also binds. The probes have a fluorescent label that is inactive as long as the probe is intact.

However, the probe does not remain intact. If, after the deposition phase, the polymerase begins to elongate, the probe is in the way. It is destroyed by the polymerase during the extension. This releases the fluorescence and the sample begins to glow.

Sources: 

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