Tue. Apr 7th, 2020

How is coronavirus detected in tests?

In the current state of alert, a useful measure to control the epidemic is to perform tests to detect the SARS-CoV-2 coronavirus. For this reason, this week the Government of Spain has announced the arrival of material for testing en masse. How does this test work? And more importantly, what are its limitations?

Taking advantage of our biochemistry

The WHO-recommended test for detecting coronavirus is called the “reverse transcriptase polymerase chain reaction”, better known by its acronym RT-PCR. This technique is not new, but has been used routinely in scientific laboratories and hospitals since its discovery in the 1980s. It is a really useful test for biologists since it allows detecting the presence of a specific DNA fragment, taking advantage of the own biochemical reactions that occur inside our cells.

To do the procedure, a sample of saliva and mucosa is removed from the patient we want to test. Our objective will be to determine if the virus is present in those few milliliters of sample. To reduce the possibility of contagion through the sample, it is subjected to extreme temperatures and chemical solutions that break the possible virus into the proteins and nucleic acids that compose it. This deactivated virus is no longer contagious, however, these tests are preferably carried out in laboratories with biological safety mechanisms that prevent the researcher from ever coming into direct contact with the sample and with any possible surviving virus.

The RT-PCR test is able to detect a DNA sequence in the sample, but the problem is that the SARS-CoV-2 coronavirus does not have DNA inside it, but RNA, another type of nucleic acid. For this reason, a previous step is necessary that we can only do thanks, paradoxically, to our knowledge of viruses. Some viruses have an enzyme called reverse transcriptase polymerase, capable of passing RNA into DNA. We are currently able to synthesize this enzyme and use it in the sample to pass the RNA from the possible coronavirus into a DNA sequence that we can use. This step is what gives that RT to the name of RT-PCR.

The next step is to search for the coronavirus with the PCR process. To do this we take advantage of the action of polymerase, an enzyme present in all living things and that has the ability to make a copy of DNA. In the cells of living things, this duplication is what allows cells to divide and have an identical copy of DNA in each daughter cell.

But for the polymerase to work, one requirement needs to be met: There must be two small RNA sequences called primers nearby. One primer indicates to the polymerase where part of the DNA should start duplication, while the other primer indicates the end of the process.

In a normal cell, there are other proteins that generate primers to duplicate all the DNA, but in a test tube, things change, and we are free to introduce the primers that we want, controlling which fragment of DNA we are going to copy exclusively. . In hospital laboratories, there are specific primers that target DNA fragments unique to viruses and pathogens. In this way, the polymerase reaction will only duplicate the DNA of those sequences if the primers find them. If the sequence, and therefore the pathogen, does not exist; the polymerase will do nothing, and the DNA will remain unduplicated.

This duplication of DNA does not occur once, but several times. Each time the polymerase makes a copy with the primers, the amount of DNA within the sample is doubled. In this way, a small DNA strand of a virus can multiply to hundreds and thousands of copies. To do this, a thermocycler is used, a small laboratory machine that subjects the sample to different temperatures for different times, in a very similar way to a kitchen robot. The polymerase only works at an ideal temperature, so by changing the temperature the thermal cycler can turn the polymerase on and off several times, leaving enough time to make more and more copies.

As a last step, the sample is stained with a fluorescent dye that stains the DNA and lets us know how much there is. If the sample has much more DNA than at the beginning, it is because the polymerase has been able to multiply the DNA of the virus, and we say that the test was positive. If the amount of DNA has not changed during the test, it is that the polymerase could not recognize the viral sequence and the test is negative.

The test is not excessively complicated to perform by someone with previous experience. There are many models of thermocycler on the market, but most are capable of processing up to fifty samples at the same time, requiring only a few hours to wait for results. Reagents such as polymerase or fluorescent dye are already mass produced for laboratories around the world, since they do not depend on what we are detecting.

The biggest problem with the test for the coronavirus and that caused a delay in having it ready were the primers, the sequences that mark the coronavirus. Like the rest of viruses, SARS-CoV-2 has a mutation rate, so your RNA changes over time. If we use a primer from one of these variable regions, there is a danger that the RT-PCR test will have false negatives for not adequately recognizing the virus.

In this sense, the investigations of the last months about the weak points of the virus They have helped generate better screening tests. Now we know that protein S, a protein necessary for virus invasion, it is stable over time. This makes it an ideal weak point for generating a vaccine and at the same time it is perfect for the RT-PCR test to use the related RNA sequence as primers.

The government has gotten more thermal cyclists and more reagents to start testing more massively, but we must be aware of the limitations of it. It all depends on when we extract the sample from the patient. If the infection is too early, the virus may not yet be present in the patient’s mucosa and the test will be negative. Nor will you see anything in people who have already overcome the disease, precisely because they have expelled the virus from their body.

In this sense, the test only works in people with a high viral load and who are currently undergoing the infection process. Something ideal for hospitals but little useful to know who has already gone through the disease.

Looking for the consequences instead of the cause

Parallel to RT-PCR, the scientific community has developed other tests that may be useful in detecting the coronavirus. The one that promises the best results in the short term is the serological test, carried out with blood samples from the patient. The main difference with RT-PCR is that in this test we did not look for the virus, but for the antibodies that we have developed against it.

Once we are infected, our immune system generates antibodies that specifically bind to the virus to mark and destroy it. These antibodies remain in our blood even after overcoming the infection, being responsible for our not becoming infected again. In serological tests, antibodies are removed from the blood sample and exposed to virus proteins, checking to see if any antibodies bind to the virus. This union implies that the antibody has been generated to recognize the coronavirus itself, therefore it is concluded that the patient has already fought or is fighting against the disease.

This test is faster and does not require so many intermediate steps, so it is possible to do it in twenty minutes. Last week a team from the Massachusetts Institute of Technology showed a prototype of strips that allow the serological test to be done quickly and safely, which would be perfect to be carried out in emergency situations like the current one.

However, when compared to RT-PCR it has its own limitations. Our antibodies can be very varied and they may recognize the coronavirus by chance. It is precisely this variety that helps the immune system to act early against unknown threats. In serological testing this is a problem since random recognition means that a small number of tests can give false positives.

The other limitation of this test is that it requires that we have a strong enough immune response against the virus, and this does not happen until a week after being infected. For this reason, serological tests are not useful to detect if someone has the coronavirus at the same time, but rather serve to know if someone has already had it and to be able to track down possible threats in people at risk.

Better strategies can be carried out in managing the pandemic if we know who has already gone through the disease, even asymptomatically, and therefore if they are immune to it. Both tests have their strengths and weaknesses, the important thing is to know their limitations and know who to apply them to.


  • The RT-PCR test only tests positive in a certain time window. If someone thinks they have just been infected or have already passed the disease, they will test negative.
  • Thanks to this test, it has been seen that the virus is also present in faeces and mucosa, which is why those infected or suspected to be infected should wear a mask and especially control their hygiene.



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