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Efficient PCR tests

"More than 4,000 samples in 24 hours"

The device from PerkinElmer chemagen Technologie GmbH is based on a KIT patent for the manipulation of magnetic particles. (Image: IFG, KIT)

Researchers from Karlsruhe have been able to make PCR tests for detecting coronavirus infection more efficient. Their invention has become established in practice: Devices based on their principle have a market share of around 25 percent in Germany alone.

The PCR test is currently the most reliable method for detecting infection with SARS-CoV-2. Fragments of the virus' genetic material are detected and multiplied. Before this can be done, however, the strands of genetic material contained in the smear must be purified and concentrated. An invention by scientists at the Institute for Functional Interfaces at the Karlsruhe Institute of Technology (KIT) makes this procedure more efficient. Matthias Franzreb and Christof Wöll explain how it works.

The conventional PCR test explained step by step: What happens to the rod after the oral-nasal swab has been taken?

Matthias Franzreb: The first step is the purification of the RNA: The swab is dipped into a solution in which the biological material, i.e. mucosal cells and possibly also virus particles, are broken up. This releases the RNA - the genetic material of the viruses, if present - and it floats in the solution. Now magnetic particles are added, to whose surface DNA or RNA selectively binds, while cell proteins, for example, remain in the solution. Now I have to separate the particles with the bound RNA from the impurities. The usual step is to hold a magnet to it from the outside. This collects the particles on the outer wall of the container. Finally, I can pull off the impurities and put in a new solution. The magnetic particle-RNA connection is separated again and I have the genetic material in a purified form. Only now can the actual PCR test start. This would not work in the original solution because of the many impurities.

Matthias Franzreb heads the Department of Bioprocess Engineering and Biosystems at the Institute of Functional Interfaces at KIT. (Image: private)

Why does the RNA strand of the virus have to be amplified at all? If I have the strand, I already know that there is a virus.

Matthias Franzreb: Only about one millionth of a gram of genetic material can be obtained from the smear. Moreover, even with positive samples, only a small proportion of this consists of viral RNA. The crucial thing about the PCR method is that these tiny amounts are selectively amplified and thus made detectable. Whether I want to detect the virus via a luminous dye or as a colored band of a gel, I simply need a certain amount of its genetic material. Here comes the additional difficulty: SARS-CoV-2 is an RNA virus and the amplification is only possible with DNA, i.e. one must first transcribe the RNA of the virus into DNA with the help of enzymes.

So much for the conventional method. With your invention, you have managed to make the sample preparation of the PCR test more efficient. How exactly?

Matthias Franzreb: The method of holding a magnet to the vessel from the outside works quite well manually, but is difficult to automate.

Christof Wöll: You can do that if you have maybe only ten samples. Now we have many tens of thousands per day. So this procedure would not be applicable at all to be able to handle all cases.

Matthias Franzreb: We wanted to parallelize the whole process, speed it up, and integrate the multiple mixing of magnetic particles with new solutions. In the classic methods, you pipette the solution, so you suck out the old solution and put in a new solution. But the particles always stay in the vessel. We had the idea that we don't transport solutions, but the particles.

And how does that work?

Matthias Franzreb: With a soft magnetic needle, a metal rod that can be made into a magnet from the outside. It collects all the particles, then I can slowly pull the rod up and change the solution. For the subsequent mixing, my colleague Jonas Wohlgemuth had the crucial idea: The needles rotate very quickly around their own axis. And if I magnetize contactlessly from the outside, I can also do this with an entire array of samples, with 96 pieces in 30 minutes. Automated like this, it's much more reproducible and faster than if someone has to do it by hand. If the genetic material of the virus is present, it is isolated and then ready for PCR. One device can thus prepare more than 4,000 samples in 24 hours.

The procedure has already proven successful for HIV and hepatitis tests in blood donations?

Matthias Franzreb: Exactly. It's the same molecular diagnostics. Also in BSE tests - that's why the market share in England is particularly high. There, the tests have made it possible to select the sheep population in such a way that only those animals that are not genetically susceptible to the disease, known as "scrapie" in sheep, are allowed to reproduce. The devices based on our invention from the licensee PerkinElmer chemagen have already been on the market for some time and were already established in larger clinical analytical laboratories at the beginning of the pandemic.

Christof Wöll heads the Institute for Functional Interfaces at KIT (Photo: private)

You developed your invention in Karlsruhe. How did you get in touch with the industry, with the company PerkinElmer chemagen?

Matthias Franzreb: A colleague there recommended us as experts in applications of magnet technology for biological issues. The development via a first and second prototype to the license agreement for the construction of the devices was extremely fast.

Christof Wöll: The mission of the Helmholtz Association includes basic research on the one hand, and application, translation into commercial use, on the other. We've also done spin-offs. But here, the best way was to first apply for a patent and then grant the company a license. We see the success in the licensing revenue. Due to the pressure during the Corona pandemic to carry out as many tests as possible as quickly as possible, this method quickly achieved a 25 percent market share in Germany - in other countries, the market share is sometimes even higher.

Where do you go from here? Do you want to improve your invention even further?

Matthias Franzreb: At the moment, we are developing methods to use electric fields to make the binding process of the genetic material more efficient. We are also trying to save chemistry in the solutions. If I could replace some of the chemistry by switching an electric field on and off, that would of course be advantageous.

Christof Wöll: In the past, we optimized these processes primarily using the extremely time-consuming "trial and error" method. The new approach is an essential element of the Helmholtz research program "Materials Systems Engineering". This involves the digitization of materials research. So we first want to optimize in the computer. Because electric fields can be simulated well, this method is particularly suitable for this.

Message from KIT

Helmholtz Association: Transfer

Completion of the merger at KIT

Federal Minister of Education and Research Anja Karliczek and Baden-Württemberg's Minister of Science, Research and the Arts Theresia Bauer have agreed on further steps to complete the merger at the Karlsruhe Institute of Technology (KIT). A renowned university and a major research center are thus deepening their merger, which already took place in 2009. In order to exploit the full potential in research, teaching, and innovation, administrative hurdles will be removed and more flexibility in the use of funds will be made possible.

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