PCR technique: How to determine viruses

Coronavirus can be detected by means of the polymerase chain reaction (PCR). This method is also used at the TU Wien. ICC member Rita Linke explains how it works.

ICC-Member Rita Linke explains how the PCR technique works.

Author: Florian Aigner

Testing, testing, testing! This is one of the most important strategies in the fight against the new coronavirus SARS-CoV-2. But how does it actually work? There is much talk about the PCR technique, the "polymerase chain reaction". This is a process for the duplication of DNA.

This technology is also used at TU Wien: "At the Centre for Water and Health we use the PCR method, among other things to test water samples for traces of fecal contamination," says Rita Linke. "Today, PCR technology plays an important role in many areas of molecular biology and medicine - including coronavirus testing."

On track of DNA

There are several ways to test people for a virus disease. For example, if you find certain antibodies in the blood, you can conclude that the person has come into contact with the virus. But it takes a while for the immune system to produce antibodies. It is therefore more reliable to look for the virus itself - or more precisely: for a very specific characteristic section of its genetic material through which the virus can be clearly identified. Because a sample usually contains only a very small amount of this genetic material, it must first be amplified in order to detect it, and this is exactly what the PCR method does.

"At the beginning we have to process the sample so that the DNA strands are exposed," explains Rita Linke. "If you're looking for bacteria, for example, you first have to break open the cells and get the DNA out." With viruses, this is relatively simple: corona viruses have a shell that can be easily destroyed, and the genetic material of the virus is located directly beneath it.

However, corona viruses do not have DNA, but RNA. The most important difference is that DNA forms a double helix - like a winding rope ladder. Between the two strands, which form the "backbone" of DNA, there are cross connections - the "rungs" of the ladder. Each rung of this ladder consists of a base pair. The genome of the coronavirus, on the other hand, consists of a single strand of RNA and looks like half a DNA molecule: It has only one backbone strand to which individual bases are attached. "However, such an RNA strand can be rewritten as a DNA strand. This is possible with an enzyme called reverse transcriptase," explains Rita Linke.

Split, mark, copy

As soon as the DNA is free in the sample, the PCR procedure can be started: The first step is to heat the sample to approximately 95°C. This opens the double strand of DNA, similar to a zipper, and you get two complementary halves. Then the DNA section to be amplified must be marked. "This is done using primers," explains Rita Linke. "These primers are tailor-made to sit on a very specific section of the strand. They mark the point on the DNA strand where duplication is to begin.

Now the actual main character of the whole process comes into play: the polymerase. It is an enzyme that can replenish the separated DNA half to form a whole DNA double helix. The polymerase docks at the site of the primer and starts working right there. The molecular building blocks that are required to complete the DNA double helix "swim" around, the polymerase makes sure that they find their place, base by base. This process is very fast: the polymerase is able to process approximately a thousand bases per minute.

On the opposite half of the DNA strand, exactly the same thing happens - but in the opposite direction. There, another primer docks on, marking not the beginning but the end of the section to be amplified. The polymerase also docks there and starts to work - but from the back to the front. "The polymerase continues working base by base on both halves of the strand. The starting point, not the end point, is given on both sides. The polymerase can overshoot the target a little - but only the area that we have selected and really want to duplicate is duplicated on both sides, on both halves of the strand," explains Rita Linke.

The decisive factor is that after this step, two complete DNA strands are once again present - and now the cycle starts all over again: The sample is heated again to separate the DNA strands, the primer molecules attach themselves again and the polymerase does its job again - and now four DNA strands are already present. In the optimal case, the amount of DNA strands doubles in each step. The desired DNA sequence multiplies exponentially. "Typically, we perform about 30 to 40 such cycles," says Rita Linke.

Evidence

At the end, after a sufficient number of PCR cycles, there are two possibilities: Either the desired DNA sequence was actually present in the sample at the beginning, in which case it was exponentially amplified and is now present in large numbers and can be detected - or this DNA sequence was not present in the sample, in which case it is still not present. In classical PCR, the final detection is then carried out in a further step.

For the detection of coronavirus, however, a modern form of PCR, real-time PCR, is used. With this form of PCR, the reaction can be followed in real time by adding fluorescent dyes. The fluorescence signal generated correlates with the amount of PCR product formed. This method has the advantage that the result is immediately visible without any further processing step. This further reduces the waiting time for the result. Of course, the use of controls is important in order to secure the results obtained.

A method with many advantages

The PCR method has several important advantages: it allows the virus to be detected directly from its genetic material, only a very small amount of material is needed to detect it, and the procedure is relatively rapid, usually taking only a few hours.

The costs of such tests are also manageable: "The reagents required for this are now produced industrially," says Linke.

Due to its wide range of applications, PCR technology has become a popular standard method for many research areas. However, this does not mean that every laboratory that has PCR machines can now usefully carry out corona virus tests. "It is not quite as simple as that," says Rita Linke. "For good reason there are precise regulations: Not every laboratory is allowed to work with pathogens. Very specific safety precautions are necessary. And even if the method is a standard procedure today - you still need trained staff."

In addition, the medical sector has particularly high quality requirements. These ensure that the products, processes or services tested are reliable and safe, and that they comply with the requirements of the relevant standards, guidelines and laws. This requires accredited test procedures, which are not available everywhere.