What is test security
How does a PCR test work?
A PCR test is carried out, for example, by the family doctor or in special test centers. First, doctors or trained healthcare professionals take a sample. Usually, a swab is taken from the upper airway for the test. This usually takes the form of a mouth or nose and throat swab.
Gargling with a rinsing solution is also possible. A blood sample is atypical for a corona detection - but widespread, for example, in newborn screening.
Regardless of the type of sample taken, genetic material is found on the cotton swab, in the rinsing solution or in a drop of blood. This sample material is sent to a laboratory, where it is isolated and purified.
A PCR test is then divided into two steps:
- PCR: In this step, the amount of the original genetic material is multiplied.
- Electrophoresis: In the second step, the genome segments are "sorted" according to size. In this way, the sample can be characterized - the fine structure of the genetic material is determined.
Step 1: PCR - "The polymerase chain reaction"
The "PCR" is the first of two working steps: Here the amount of existing starting DNA is multiplied. Because only when the genetic material is available in sufficient quantity can it be examined. Mostly this is human DNA; tests for the coronavirus are accordingly virus RNA.
The abbreviation PCR stands for the English term “polymerase chain reaction”.
What is needed for a PCR?
The starting DNA is located in a reaction vessel with special substances. The existing genetic material serves as a template, which is copied in the presence of certain enzymes (Taq polymerase) and certain basic DNA building blocks.
The copying process takes place in several - repetitive - runs (cycles).
Specifically, the following substances are combined:
- Starting DNA: Sample material to be reproduced.
- Basic DNA building blocks: These are the nucleobases adenine, thymine, cytosine and guanine.
- DNA polymerase: An enzyme that links individual DNA building blocks to form a well-defined strand of DNA. The newly obtained strand is a mirrored (complementary) image of the original starting material.
- Buffer solution: The polymerase chain reaction takes place in a physiological buffer solution. This ensures conditions similar to those found in human cells. This solution keeps the pH stable and also contains magnesium ions. These controlled conditions are necessary to ensure the functionality of the DNA polymerase.
- Primer: They consist of 16 to 24 base pairs and serve as a start position and start signal. Primers show the DNA polymerase at which point (of the starting DNA) the copying process begins.
How is a PCR test done?
Since all the substances required for a PCR are now in the reaction vessel, the actual copying process of the genetic material can begin. This is only started, controlled and stopped again by the temperature.
The reaction vessel is consequently heated or cooled in a targeted manner to different temperatures. This is done with a special device - the so-called thermal cycler. The overall reaction takes about one to two hours.
The individual steps of a PCR cycle are:
- Denaturation of the DNA double strands: The sample is heated to around 90 degrees Celsius. As a result, the original DNA double strand breaks into two separate (complementary) single strands.
- Attachment of the primers: The temperature is lowered to slightly below 60 degrees Celsius. As a result, the primers (forward primers, reverse primers) attach to defined positions on the corresponding single strands of DNA.
- Renewal: The temperature is increased to slightly above 70 degrees Celsius. The DNA polymerase now begins its work in this temperature optimum. It attaches to the primer positions and extends the original single strand of DNA step by step until an exact (complementary) copy is available (DNA synthesis).
After a completed cycle, the temperature is increased again to around 90 degrees Celsius - the cycle starts all over again.
Using the PCR method, DNA sequences can be replicated up to around three kilobase pairs (kbp). This corresponds to around 3,000 basic DNA building blocks linked to form a “chain”. For comparison: the human genome stores the blueprints and information for the operation of the cell in around three billion base pairs - the coronavirus genome, on the other hand, consists of 30,000 base pairs. With a PCR test, only short sections of the total DNA can be reproduced and examined.
The primers are crucial
The choice of primers is critical to the PCR process. In Sars-CoV-2 diagnostics, for example, several primers are used (multiplex PCR).
Corona PCR tests look for three different virus genes: The overall specificity increases to almost 99.99%. This means that with this high hit rate, there is only one false-positive test per 10,000 examinations (if the sample is taken correctly).
How much copied genetic material is there now?
Let us assume that after the first cycle there are two identical DNA double strands.
After each cycle, the amount of (copied) genetic material doubles. So the amount of DNA grows exponentially.
In other words: after the second cycle there are four identical double strands - after the third cycle eight double strands - after the fourth cycle 16 double strands and after the fifth cycle already 32 double strands.
Medical professionals usually repeat this process around twenty to thirty times.
Metaphorically speaking, this means: Even if only a single DNA double strand was found in the sample at the beginning, after twenty cycles there will already be a million identical copies in the reaction vessel.
What does the Ct value mean?
The number of completed PCR cycles is given in the form of the so-called Ct value. “Ct” is derived from the English term “cycle threshold”. This Ct value enables statements to be made about the amount of genetic material being sought.
At a low Ct value of 20, there is a lot of starting genetic material. However, if the Ct value is high - around 30 cycles - there is correspondingly little DNA. The PCR cycle must therefore be run through more often.
Step 2: Electrophoresis "Sorting by Size"
Finally, if there is sufficient “enriched” genetic material, what is known as electrophoresis can take place. Scientists use a certain property of DNA: its electrical charge.
The individual DNA building blocks are linked to one another via a (negatively) charged sugar-phosphate backbone. The longer a certain DNA sequence is, the higher its electrical charge.
If the genetic material is placed in a specific carrier medium (agarose) and an electrical voltage is applied, the DNA segments begin to move due to their charge. Since a longer stretch of DNA travels faster than a shorter fragment, they separate according to size.
In this way, the genetic material can be examined and characterized. In practice, an unknown sample with a known reference is usually applied to a "starting line" and compared with one another after a certain period of time.
If the "migration speed" is the same for both sequences, this means: The detection is very likely positive - the gene you are looking for is contained in the sample.
Coronavirus special case: sample preparation and RT-PCR
The pathogen detection for the coronavirus is a special case. Sars-CoV-2 is one of the so-called RNA viruses. This means that the Sars-CoV-2 genome is in the form of RNA (ribonucleic acid).
RNA differs from DNA in only a few ways. Among other things, it is available as a single strand and is based on the sugar ribose instead of 2'-deoxyribose. The nucleobase thymine has also been replaced by uracil as the fourth base.
This viral RNA must be "transcribed" into DNA before a regular PCR test. This process is called reverse transcription (RT) - hence the term RT-PCR. In the course of this process, a single cDNA strand (“complementary DNA”) is obtained. In a further step, the single cDNA strand is supplemented by a second, mirror-inverted DNA strand.
At the end of the sample preparation, there is a DNA double strand that corresponds to the original blueprint of the virus RNA: Only this double strand can now be used as a template in a PCR test.
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