COVID-19 is an infectious disease, characterized by severe acute respiratory syndrome, caused by the SARS-Cov-2 virus. In particular, individuals suffering from this pathology present symptoms such as fever, cough and shortness of breath.
COVID-19 Diagnosis
The diagnosis of COVID-19, in case of suspicious symptoms, is made by collecting a sample from a nasopharyngeal or oropharyngeal swab.
Nasopharyngeal swab
The swab is inserted deep enough into the nasal cavity, then it is gently moved forward to the nasopharynx, rotating it for some time in order to collect any secretions possibly containing the virus.
Subsequently, it is placed in a sterile medium, a buffer (obviously, without any PCR inhibitors) and sent to the laboratory.
PCR
The standard method for the detection of Sars-Cov-2 uses a normal PCR reaction.
Specifically, we refer to a technique widely used in molecular biology that allows obtaining multiple copies of a specific DNA fragment. In particular, the Coronavirus is a very long single-stranded RNA virus and its identification occurs precisely by searching for viral RNA. Therefore, an RT-PCR is used (in this case, first the Reverse Transcriptase PCR and then the Real-time PCR). In this way, the viral RNA is initially converted into cDNA, DNA complementary to the viral RNA, through the reverse transcriptase enzyme. The product of this reaction will be an RNA / DNA hybrid. After the removal of the viral RNA, the cDNA will be used for amplification by classical PCR. However, before proceeding with the two PCR techniques, the viral RNA is subjected to extraction and purification.
Extraction
First, the sample is placed in a micro-centrifuge tube and then mixed with a lysis buffer (highly denaturing solution, containing reagents capable of easily lysing the surface of the virus). Once the buffer has been inserted into the sample and placed all in a test tube, the contents of the latter are mixed using a vortex and then incubated at room temperature.
Purification
This process takes place using a spin column (a purification column containing a specific resin).
In this column there is:
- a stationary phase consisting of a resin;
- a silica gel matrix (at optimal pH conditions for viral RNA). It strongly holds the RNA molecules.
In this way, the spin column filter will bind only the RNA molecules. Following centrifugation, the column is placed in a clean tube, while the filtrate is discarded. Subsequently, a wash buffer is added to the column, thus carrying out a series of washes in such a way as to remove any trapped residues. Then, the sample is again subjected to centrifugation. By doing this, only the viral RNA bound to the spin column silica gel is obtained. After further washing, an elution buffer is added to the sample, i.e. an elution buffer, so as to determine the release of the viral RNA. At this point, the whole is subjected to centrifugation again. Now, the viral RNA has been extracted and purified.
ReverseTranscriptase PCR
At this point, the viral RNA (the primary template) will be used for reverse transcriptase PCR in order to obtain a secondary template, namely the cDNA. Then, the cDNA will be amplified by real-time PCR. Therefore, you can move on to preparing the reaction mixture for the amplification of the viral sequences by PCR.
What happens in particular?
First, a master mix is used, a ready-made solution consisting of:
- reverse transcriptase;
- triphosphate nucleosides (dNTPs);
- reverse and forward primers;
- TagMan probe and DNA polymerase.
First, the extracted and purified RNA sample is placed in a tube containing the master mix solution and is then subjected to mixing by vortexing. The reaction mix is then loaded into a PCR plate which will then be fed into a PCR machine. Specifically, we are talking about a thermal cycler programmed first for RT-PCR and then for real-time PCR. Furthermore, this machinery is suitably set to amplify the SARS-Cov-2 target genes, that is the RdRP, E and N genes. Clearly, the choice of these genes will depend on the sequences of the primers and of the TagMan probe used.
Sequential steps
The first step operated by the device is that of retro-transcription. In particular, the synthesis of cDNA occurs thanks to the primer which hybridizes to the complementary sequence of the viral RNA and which is stretched by the reverse transcriptase enzyme, in the 5′-3 ‘direction. Subsequently, an initial denaturation is performed in such a way that the viral cDNA-RNA hybrid is denatured, the DNA polymerase is activated simultaneously and the reverse transcriptase is inactivated. Then, we move on to the action of the DNA polymerase which polymerizes the other complementary strand of the cDNA that has been retro-transcribed. After that, you can continue with the actual amplification of the double-stranded cDNA by real-time PCR.
Amplification step
The identification of viral DNA is achieved in three essential cycles, each characterized by:
1) denaturation;
2) annealing;
3) extension.
First cycle
In the first cycle, there will be the denaturation of the cDNA, the annealing (bond) between the primers of the mixture and the single helix of denatured cDNA and the extension (elongation) of the cDNA sequence thanks to the dNTPs and DNA polymerase present in the mixture. At this point, the reverse transcriptase will no longer intervene, but the Taq polymerase. Eventually, the first double-stranded target DNA molecule will be obtained (corresponding, clearly, to SARS-Cov-2 RNA).
Second cycle
In the second cycle, the denaturation of the cDNA and the annealing between the probes of the mixture and each of the two single helices of template DNA complementary to the target genes will take place. TagMan probe will also be annealed to the complementary sequence of the target DNA. In the subsequent extension phase, the DNA polymerase that will synthesize the new DNA double helices, flowing along the single strands, thanks to its exonuclease activity, will also degrade the TagMan probe. By doing so, the probe, quencher and fluorophore will be separated and the fluorophore will begin to fluorescence.
Third cycle
In the third cycle and so on in the following cycles, there will be further denaturation of the cDNA helices, further annealing between the probes and the target genes and further extension for which there will be a new synthesis of complementary fragments. In this way, in the end, we will be able to detect or not the presence of SARS-Cov-2 viral RNA.
Giovanna Spinosa translated by: Umberto Lazzaro
Sources:
- https://etd.adm.unipi.it/t/etd-09172021-123453/;
- http://www.omceopr.it/wp-content/uploads/2020/03/NOTA-SULLA-DIAGNOSTICA-COVID-E-SICUREZZA-DEI-TEST-.pdf;
- https://www.ordinefarmacistigr.it/documenti/circolarifofi/2020/12616.pdf.
Image credits:
- Figura 1: https://www.serchioindiretta.it/cronaca/2021/12/20/coronavirus-87-nuovi-positivi-in-provincia-di-lucca-8-sono-in-valle-del-serchio/132892/;
- Figura 2: https://www.regione.lombardia.it/wps/portal/istituzionale/HP/lombardia-notizie/DettaglioNews/2020/08-agosto/a/19-08-2020-aeroporto-malpensa-tamponi;
- Immagine 3: https://www.genomeup.com/Glossary/pcr/;
- Immagine 4: https://www.multimed.it/blog/blog/salute/tampone-molecolare-covid-19-tutto-quello-che-c-e-da-sapere.
