Article Writer- Om Bikash Sahu, Int. MSc., School of Biological Sciences, NISER, Odisha(India)
Different methods of testing methods are experimented in different countries to trace COVID-19 infection. Among them the most accepted and wildly used test is RT-PCR. In India, the health ministry has announced that the RT-PCR technique is the gold standard frontline test for the COVID-19.It is a direct method of testing in which it detects the presence of the virus in the affected area.
Methodology for the RT-PCR
1.Sample collection –
We have to collect a sample to perform the test. Technicians enter a cotton bead inside the nose or mouth to extract swab (nasopharyngeal swab or oropharyngeal swab) from it because if COVID-19 virus infection occurs then we can able to find traces of virus there easily. After the collection of samples, they should keep it in a viral transport medium to safely transport the sample.
2. RNA collection and purification –
we have to first isolate the RNA from the sample by using different methods. To so this at first, we have to add some lysis buffer (used to denature all biomolecules except RNA) which contains phenol and guanidine isothiocyanate into the sample. The lysis buffer also includes RNAase inhibitors to protect RNA from degradation. By properly mixing the lysis buffer we can lyse all biomolecules except RNA. Now for the purification and isolation of RNA technicians generally used spin column followed by centrifugation. In this solid-phase extraction technique, RNA can able binds to the silica membrane whereas other biomolecules cannot. After the centrifugation, the filtrate should be discarded and the spin column is transferred to other sterilized microcentrifuge tubes. By using wash buffer and centrifugation we can able to eliminate all remaining lysed biomolecules. By using the elution buffer and centrifugation process, we can collect the pure RNA from the silica column.
This is a combination of 2 steps. 1. Reverse transcription 2. PCR. a reaction mixture is prepared for this step which contains a buffer, reverse transcriptase dNTPs, reverse primer, forward primer TaqMan probe, DNA polymerase, and RNA templates which is added to the purified RNA. Then placed it in the machine.
In this step, we can produce DNA from RNA by using a reverse transcriptase enzyme. For this, we have to know the sequence of the gene of the virus which is provided on the website. Ex- RdRP gene, E gene, N gene sequences. According to the gene sequences we have to design and use a reverse primer which helps to initiate the DNA synthesizing process. The enzyme reverse transcriptase is used for the addition of dNTPs for production of complementary DNA. The primers are always added to the three prime ends.
The reverse transcription reaction is followed by PCR by which the amount of complementary DNA increases. . PCR comprises of 3 stage 1.heat denaturation 2. Annealing 3. Extension
In the first cycle of PCR. After the production of the complementary DNA initial denaturation occurs which helps in the inactivation of reverse transcriptase and activation of the Taq polymerase enzyme. In the annealing part forward primer is added to the 3’ end of the cDNA. In the extension step, the polymerase enzyme helps the production of the double-stranded DNA.
In the second cycle of PCR, in the denaturation step, the ds DNA breaks into single-stranded DNA. In the annealing step, the primers to the target sequence and also the TaqMan probes are attached to the target sigh which contains the fluorophore covalently attached to the 5’ end of the probe. There is a quencher that is attached to the 3’ end which helps in the specificity of detection. These fluorophores and the quencher help in the detection. In the extension step, the fluorophores are removed by the exonuclease activity.
4.Detection by the picture and the graph
By the repetition of the cycle in PCR results for the production of more DNA and more fluorescent dye is produced so that it results in the positive result of the COVID-19 test. We can also able to get the result from the graph which is drawn in the computer.
Advantages of RT-PCR
No post PCR processing required
Amplification can be monitored in real-time
Disadvantages of RT-PCR
High time consuming
High money consuming
Well trained technicians required