Two years after the outbreak of the pandemic in Spain, more than 45 million PCRs have been performed. This is an unprecedented figure for this laboratory technique that has been crucial after the appearance of the coronavirus. However, this test is not new. On the contrary, it has been used for more than 30 years and in the last 20 it has become a basic technique in microbiology laboratories. "Long before COVID-19, there were commercial PCR methods against various viruses. The set-up for SARS-CoV-2 has been the usual for any PCR-based diagnostic kit," explains Juan Echavarría, a researcher at the Center National Institute of Microbiology of the Carlos III Health Institute.
In total in Spain, more than 77.6 million diagnostic tests between PCR and antigens have been carried out to date. The last disaggregated data provided by the Ministry of Health it is from the month of november, at which time 62.2 million diagnostic tests, 44 million PCR tests and 18.2 antigen tests had been carried out. In that month, some 300,000 weekly PCR tests were being carried out. It has not been a coincidence that in such a short time it has been possible to carry out such a large number of PCR tests for the new infection, indicates Echevarría. All the hospitals in Spain already had the necessary instruments to carry them out.
Estrella knows well the story behind the diagnostic kits for SARS-CoV-2. In March 2020, this biotechnologist worked in a private laboratory that focused all its efforts on developing the test as quickly as possible. "The normal developments of a company, from the time a need is identified until a diagnostic kit is launched on the market, can take more than six months. Here it took us a month and also for something for which there was not much information yet", recounts.
Reagents are used throughout the process that were very scarce in March 2020. "This was one of the problems. When the pandemic occurred, there were very few companies in Spain with the capacity to produce reagents. We had absolute dependence on foreign countries," says Echevarría. The same thing happened with reagents as with masks or respirators. In the wake of the pandemic, there are now more companies "capable of producing them." "When the pandemic passes, it must be a strategic objective to maintain that capacity to produce reagents," adds the virologist.
PCR is a molecular biology technique developed in 1986 by Kary Mullis, an American biochemist who won the Nobel Prize in Chemistry – shared with Michael Smith – for polymerase chain reaction (PCR). The test essentially consists of amplifying a fragment of the genetic material from the patient's sample to find out whether or not it has genetic material from the SARS-CoV-2 virus. It is replicated until enough is achieved to analyze it and for the result to be reliable. It is a snapshot at a given time that allows you to see if a person has the infection at that time.
The first thing to do is obtain the clinical sample. To do this, health personnel insert a swab through the patient's nasopharyngeal or oropharyngeal route and take the sample. The swab is inserted into a tube that is identified with a code. Inside, the tube has a liquid that stabilizes the sample and preserves it.
The next thing to do is to inactivate the sample. Scientists cancel the contagious capacity of the virus by adding another liquid, but the genetic material remains. After this, the genetic material is extracted from the sample. The sample contains cells from the individual (with their proteins, DNA, and RNA), as well as viral RNA and protein (if the person is infected). This is a highly automated process that is done with laboratory equipment. Through reverse transcription, the viral RNA in the sample is converted into complementary double-stranded DNA strands, which is the form that the genetic material must have in order to perform the PCR.
This leads to amplification. When the extract of the genetic material is already available, the actual PCR technique is performed. Some reagents are added to the sample along with an enzyme that, through cycles of temperature changes, makes millions of copies of the virus's genetic material. At the end of the process, which is carried out with a thermal cycler, a graph is generated on a computer that shows or not the presence of the virus and in what quantity. It is something like a real-time photograph of a sample at that moment.
But PCR is not only used to detect infectious diseases. It is also used in criminology, in paleontological studies or to detect food fraud. "In criminology it is used when you want to know who a biological remains belongs to," Echevarría exemplifies. It is amplified by PCR for later sequencing. Finally, it is compared in a database."
If there are doubts, it can also be used to find out if certain products intended for consumption "are of a certain species" or they are not. "A variety of things. If we are told that a rug is Persian, we can find out by amplifying the genome of the animal from which the wool was obtained." PCRs work with old samples. In fact, "that is what has made it possible, for example, to have the Neanderthal genome," says the virologist. Even if thousands of years pass, if the storage conditions are good, it is possible to amplify the DNA with a small sample.
"It was a very overwhelming time, but few times in my life have I been as motivated as when I worked on this," recalls Estrella. "Every scientist wants what he is investigating to bear fruit and it motivated me a lot to know that we were doing something that was going to serve society. Hospitals needed PCRs."
Echevarría acknowledges that the role of scientists in laboratories has been "enhanced" with the pandemic. He considers that they are workers who are "behind the scenes", but who make "things work". "We have done more than one laboratory test for the same agent per inhabitant. The numbers are not comparable with anything. We are in a completely different magnitude in terms of the number of PCR tests."
The data is "unprecedented", insists the researcher, who adds that in this time five million complete genomes of SARS-CoV-2 have been sequenced worldwide. "Before the pandemic, the virus that had the most complete genomes was HIV and there were 12,000. This is another magnitude and has nothing to do with the magnitude of the diagnostic effort," he concludes.