Performance of PCR for detection of COVID-19

The SARS-CoV-2 virus is a simple positive-sense RNA virus belonging to the Coronavirus family. This virus is responsible for the COVID-19 disease, declared a pandemic in March 2020 by the World Health Organization. COVID-19 is a disease that fundamentally affects the respiratory tract, giving rise to symptoms such as a dry cough or fever, and in the most severe cases, bilateral pneumonia that sometimes leads to the death of the patient. 

The special importance of the SARS-CoV-2 virus lies in its transmission speed or R0, since it has an R0=2-6.47, which indicates that each infected person can transmit the virus to between 2 and 6.47 healthy people, through the droplets that are released when sneezing, coughing or speaking, favouring its rapid dissemination among the population. Thus, in order to contain transmission, in addition to adopting health measures, effective detection methods must be available, which allow people carrying the virus (infected) to be located and isolated from the rest of the population.

What is RT-PCR?

One of these methods is the Real Time Reverse Transcription Polymerase Chain Reaction (RT-PCR), which aims to detect active SARS-CoV-2 infection.

The RT-PCR for the detection of SARS-CoV-2 is a technique that detects the genetic material of the virus, RNA, specifically from a patient’s nasopharyngeal or saliva sample. In short, the technique uses fluorophore-labelled probes that bind specifically to specific sequences of the virus RNA, allowing the amplification of the RNA and its quantification by fluorometric methods.

However, for the RT-PCR to be successful, the virus RNA must be previously isolated from the sample, a process that generally includes the following steps: cell lysis; separation of RNA from other macromolecules such as DNA, proteins, and lipids; and concentration of the extracted RNA.

How is the test performed?

For the isolation of RNA, a nasopharyngeal or saliva sample contained in a 1.5 ml tube is used. After cell lysis, the RNA is extracted by precipitation with ethanol, and the sample is washed with different washing buffers. For this step, the lysed sample is placed in an extraction column attached to a 2 ml capacity collection tube, and it is centrifuged for 1 minute at 8,000 rpm each time a new reagent is added. In addition, it is recommended to carry out an additional centrifugation step of 10 minutes at 13,000 rpm at the end, to remove all the ethanol from the sample, as this is a strong inhibitor of RT-PCR. 

Finally, the RNA contained in the column is eluted, using an elution buffer, and centrifuging the sample again for 1 minute at 8,000 rpm. Once the RNA has been extracted, the RT-PCR is performed, following the manufacturer’s instructions according to each case.

To carry out all these necessary centrifugation steps we can rely on the microcentrifuge Biocen 22. With a rotor for 24 samples of 1.5-2 ml, it can be programmed in a working range from 100 to 15,000 rpm. It also has a function that allows the equipment to be programmed with countdown/up from “0” or at “set RPM/RCF”, which is very useful for this type of procedures that require reproducible test results.

RT-PCR, one of the most widely used methods for the detection of SARS-CoV-2, requires the extraction of RNA from the sample as a first step, and when this extraction is carried out by means of ethanol precipitation, the centrifugation steps are fundamental.