What is a PCR test?
A PCR test is a laboratory method used in molecular biology and medicine. The test is used for the direct detection – and characterization – of genetic material. The PCR method is considered by experts to be easy to perform, universally applicable and robust.
In the laboratory, a PCR test consists of two steps. In the first step, the existing genetic material is amplified using the polymerase chain reaction (PCR). This allows the smallest traces of DNA to be examined. This is also the reason why PCR tests react so sensitively.
In a second step, the genetic material is separated according to its properties, “sorted” and thus characterized. In other words, the fine structure of the DNA is determined.
There are many possible applications: doctors use a PCR test, for example, to examine swabs for the presence of coronaviruses, blood donations for HIV or to screen newborns for possible hereditary diseases. Bacterial infections – for example with the tuberculosis pathogen – or parasitic infections (malaria) can also be clarified using PCR.
They also help to convict perpetrators in forensic medicine on the basis of their genetic fingerprints or are used as paternity tests.
How long is a PCR test valid?
A PCR test result is usually valid for 48 hours from the time the sample is taken.
How reliable is a PCR test?
PCR is a tried and tested detection method in molecular diagnostics and medicine. It is regarded as the so-called gold standard with a very low error rate. The tests are characterized in particular by a very high sensitivity and specificity.
Sensitivity means the reliability with which a test finds the genetic material to be detected.
Specificity means the certainty with which the test determines that the genetic material in question is not present in the sample.
When do PCR tests work for a Sars-CoV-2 infection?
As a rule, the PCR test detects a corona infection two to three days before and up to 20 days after the onset of symptoms. Even in infected people who remain completely symptom-free, the test is effective in a critical time window when they can infect others.
In individual cases, detection is even possible 60 days after the onset of symptoms.
Possible sources of error
The error rate in the DNA copying process is negligible in practice. Although DNA polymerases are never error-free, they hardly play a role in the PCR test procedure.
In practice, potential sources of error lie more in the sample collection: it is therefore important that trained medical professionals carry out the swabs. Saliva and gargle samples could possibly reduce the accuracy of the test, as dilution effects occur here.
How does a PCR test work?
A PCR test is carried out, for example, by a family doctor or in special test centers. First, doctors or trained medical professionals take a sample. A swab is usually taken from the upper respiratory tract for the test. This usually takes the form of a mouth or nasopharyngeal swab.
Gargling with a rinsing solution is also possible. A blood sample is atypical for corona detection – but widely used in newborn screening, for example.
Regardless of the type of sample taken, genetic material is found on the cotton swab, in the rinse solution or in the 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 quantity of the initial genetic material is amplified.
Step 1: PCR – “The polymerase chain reaction”
The “PCR” is the first of two steps: This is where the amount of available starting DNA is amplified. This is because genetic material can only be analyzed once it is available in sufficient quantities. In most cases, this is human DNA; in the case of tests for coronavirus, it is viral RNA.
The abbreviation PCR stands for “polymerase chain reaction”.
What is needed for a PCR?
The starting DNA is placed 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 added together:
- Starting DNA: Sample material that is to be copied.
- 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 DNA strand. The newly obtained strand is a mirror image (complementary) of the original starting material.
- Primers: They consist of 16 to 24 base pairs and serve as a starting position and starting signal. Primers show the DNA polymerase at which position (of the starting DNA) the copying process begins.
How is a PCR test carried out?
Now that all the substances required for a PCR are present in the reaction vessel, the actual copying process of the genetic material can begin. This is started, controlled and stopped again solely by the temperature.
The reaction vessel is therefore heated or cooled to different temperatures one after the other. This is done using a special device known as a thermal cycler. The entire 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. This separates the original DNA double strand into two individual (complementary) single strands.
- Attachment of the primers: The temperature is lowered to slightly below 60 degrees Celsius. This causes the primers (forward primer, reverse primer) to attach to defined positions on the corresponding individual DNA strands.
After a completed cycle, the temperature is raised again to around 90 degrees Celsius – the cycle starts again from the beginning.
The PCR method can be used to amplify DNA sequences of up to around three kilobase pairs (kbp). This corresponds to around 3,000 basic DNA building blocks linked together 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. A PCR test can therefore only ever amplify and examine short sections of the total DNA.
The primers are crucial
The choice of primers is crucial for the PCR procedure. In Sars-CoV-2 diagnostics, for example, several primers are used (multiplex PCR).
Corona PCR tests thus search for three different virus genes: This increases the overall specificity to almost 99.99%. This means that with this high hit rate, there is only one false-positive test per 10,000 tests (if samples are taken correctly).
How much copied genetic material is now available?
Let us assume that two identical DNA double strands are present after the first cycle.
After each cycle, the amount of (copied) genetic material doubles. The amount of DNA therefore grows exponentially.
Doctors usually repeat this process around twenty to thirty times.
Figuratively speaking, this means that even if only a single DNA double strand is found in the sample at the beginning, after twenty cycles there are already a million identical copies in the reaction vessel.
What does the Ct value mean?
The number of PCR cycles run is indicated in the form of the so-called Ct value. “Ct” is derived from the English term “cycle threshold”. This Ct value makes it possible to make statements about the amount of genetic material being searched for.
With 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 more often.
Step 2: Electrophoresis “Sorting by size”
Once sufficient “enriched” genetic material is available, electrophoresis can be carried out. In this process, scientists exploit a specific property of DNA: its electrical charge.
The individual DNA building blocks are linked to each other via a (negatively) charged sugar-phosphate backbone. The longer a particular DNA sequence is, the higher its electrical charge.
This allows the genetic material to be examined and characterized. In practice, an unknown sample is usually plotted against a known reference on a “starting line” and compared with each other after a certain period of time.
If the “migration speed” is the same for both sequences, this means that the detection is most likely positive: The detection is very likely positive – the gene you are looking for is contained in the sample.
Special case of coronavirus: sample preparation and RT-PCR
The detection of the coronavirus is a special case. Sars-CoV-2 is one of the so-called RNA viruses. This means that the Sars-CoV-2 genetic material is present in the form of RNA (ribonucleic acid).
RNA differs from DNA in only a few respects. Among other things, it is present as a single strand and is based on the sugar ribose instead of 2′-deoxyribose. The nucleobase thymine is also 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. A single strand of cDNA (“complementary DNA”) is obtained as part of this process. In a further step, the cDNA single strand is supplemented by a second, mirror-image DNA strand.
How long does it take to get the result?
Once the sample has been sent to the laboratory, you will usually receive a result within one or two working days. In test centers, which often examine the samples directly on site, it can also take just a few hours. The time span depends largely on the respective test center and its logistics.
Despite the complex work processes, modern laboratories are able to carry out PCR tests with a high throughput. Special automated devices help to carry out the tests.
Nevertheless, the PCR test is considered a comparatively “slow” but all the more reliable detection method.
What happens if the result is positive?
If the sample is taken correctly, a positive PCR test means that there is a very high probability that the person tested is infected with Sars-CoV-2.
If you have been confirmed to be infected with the coronavirus using a PCR test, the public health department will receive notification of the positive test result from the relevant laboratory. In such a case, the public health department will order isolation or quarantine.
Am I automatically contagious if I test positive for PCR?
Usually yes. But not always. A PCR test result should always be interpreted in context. A positive test means first and foremost that you are carrying viral material.
A supplementary antibody test is sometimes useful
In such cases, an antibody test provides certainty that confirms the validity of the PCR test. It is best to discuss this with your doctor. They can help you interpret the PCR test result correctly.
What to do if the result is negative?
A negative PCR test result most likely means that you did not have Covid-19 at the time the sample was taken and are therefore not currently infectious. However, you could be in an early infection phase.
Corona infections can usually only be detected from the second or third day after infection. The result is therefore not a free pass. You should therefore continue to observe social distancing and hygiene rules and continue to wear an FFP2 mask – for your own protection and that of others.
PCR test for children
A PCR test for children is no different from a PCR test for adults. Both the sample collection and the interpretation of the results apply to children as well as adults.
What are the risks of a PCR test?
A PCR test does not involve any physical risks. Only the collection of the sample by nasopharyngeal swab is perceived by some people as disturbing or unpleasant.
What does a PCR test cost?
Please note: If you have tested yourself at home and received a positive result, you should make an appointment with your GP immediately by telephone. Your doctor will discuss the next steps with you over the phone.
Alternatively, it is best to call 116 117 to register for a PCR test. Ideally, you should stay at home until the confirmation test to protect yourself and others.
