How does a PCR test work?

Olivia Berger, a student at MCI, works in a PCR laboratory alongside her studies. We asked the Biotechnology and Food Engineering student about her work.

Ms. Berger, you have been studying in the Biotechnology and Food Engineering bachelor's program here at MCI since 2018. Now you work in a microbiology lab alongside your studies, evaluating PCR tests. Which subjects in your studies were particularly helpful for your current job?

It already starts with the basics of biology to get a general understanding of biological processes. All biological subjects (microbiology, molecular cell biology, ...) are very important for working in the laboratory. Actually, you can exclude few subjects that are not helpful, because it is usually an interaction of several fields. Biochemistry for example helps to explain processes or not expected products and thermodynamics to optimize certain processes.

You work in the laboratory "Pathologielabor Dr. Obrist Dr. Brunnhuber OG" in Zams. What is a working day like for you at the moment?

I am employed in the microbiology department. This is where the Covid 19 tests are carried out using PCR. My working day starts either at 6 AM or at 2 PM. My main task is to work with my colleague on all SARS-CoV2 samples within a very short time.

We work with different tools to be able to process several runs (one run consists of 94 samples) at the same time. The fastest method is manual extraction. Here, the extraction of the viral RNA is performed manually using centrifuges and various buffers.

Likewise, there are several automated machines in our laboratory that can perform this extraction independently.

Once the extraction is complete, RT-PCR is performed. Here, the RNA we are looking for, usually the N gene of SARS-CoV2, is amplified. If there was RNA of the virus in the sample, it can thus be detected.

At the moment, the term PCR test is a very popular topic. What does PCR actually stand for?

PCR stands for "polymerase chain reaction" and can be used to amplify DNA. Actually, in the SARS-CoV2 tests, an RT-PCR, i.e. a so-called real time PCR, is performed. The difference is that RT-PCR can measure the amount of PCR product in real time during amplification by fluorescent dye.

Initially, the obtained sample of RNA and various reagents are pipetted together into a PCR plate. By adding the enzyme reverse transcriptase, the RNA is transcribed into DNA. Only in this way can it be amplified by RT-PCR.

The instrument initially heats to 94°C to separate the double-stranded DNA into two single strands.  The temperature is then lowered to reach the optimal temperature for the primers. These bind to the single strands of DNA. The temperature is then raised to the working temperature of 72°C of the enzyme DNA polymerase. This enzyme synthesizes new DNA strands. This is followed by the first step again with heating to 94°C. These steps are repeated up to 45 times in our laboratory.

The DNA strands formed are marked with a fluorescent dye and can thus be detected.

And how exactly is it determined whether a sample is negative or positive?

If a sample is positive, the amplified, fluorescently labeled DNA strands of the gene are detected by a signal. If newly formed DNA strands are formed and measured early in a sample, the signal becomes stronger with each pass. This results from the increase in DNA strands per cycle. The notorious ct value is also read from these signals. This indicates after how many cycles enough DNA has been amplified (formerly RNA of the virus) to be detected. The smaller the ct value, the faster the sought-after piece of DNA was detected and the higher the potential for infection. If a ct value is above 35, then the risk of infection is no longer given and the sample is given out as negative. All samples below a value of 35 are visually checked with a 4-eyes principle to ensure that the curve represents a significant progression.

If a sample is negative, then no RNA is processed and therefore there is no detection.

Of course, we always have control samples that are tested along with each run. The positive sample consists of the gene to be detected and must always show positive. The negative sample consists of the starting reagents only without the sample and must always read negative. If one of these controls is not 100% conclusive, the run is not released and must be repeated completely.

How many samples per day are evaluated in the laboratory?

In the summer of 2020, we peaked at 3000 to 5000 samples per day. Today it's still around 1000 to 2000 samples a day.

What is the biggest challenge for you in this job?

The biggest challenge is to manage the work properly. A sample from a patient in the hospital has a higher priority than a sample from the tourism industry. And yet both samples have to be processed as quickly as possible and with the utmost care.

For me, it is very important in this work not to forget that there is a human being behind every sample number.