How COVID-19 test is done?
How COVID-19 test is done?
The sample is collected from a patient’s nose or mouth. The nasopharyngeal swab is used to collect secretions that contain viruses. The swab is then transferred to the sterile container containing a viral transport medium.
Coronavirus contains a single-stranded RNA genome
The RNA genome of coronavirus needs to be converted into their complementary DNA sequences for its testing which is done by reverse transcriptase. Then the newly synthesized DNA sequences are amplified using polymerase chain reaction. This is known as reverse transcriptase-polymerase chain reaction (RT PCR)
Real-time RT–PCR is a nuclear-derived method is used for the detection of specific genetic material in any pathogen including virus.
Corona Virus (Corona viral Disease 2019)
It is a severe respiratory disease, most common symptoms include fever, cough and shortness of breath.
How RT PCR is conducted?
Extraction of viral RNA
Viral RNA has to be extracted to carry on the process of RT-PCR.
- The sample is first added into a microcentrifuge tube and then is mixed with lysis buffer
(Lysis buffer is denaturing in nature it consists of phenol and guanidine isothiocyanate)
- Then it is led to vortex for the mixing of both and then it is incubated at room temperature which leads to the denaturation of virus
- After lysis, purification procedure is carried out by using a spin column
- The sample is now loaded to the spin column and it is let for centrifugation
- In centrifugation, the stationary phase consists of a silica matrix to which RNA molecules bind
- After centrifugation, the spin column is now transferred to a clean collection tube and the filtrate is discarded
- Then wash buffer is added to the sample
- The column is again centrifuged forcing the wash buffer through the membrane
(This removes only remaining impurities from the membrane leaving the RNA which is bound to the silica gel)
- Once it is washed again the spin column is placed in a clean microcentrifuge tube where elution buffer is added.
- Then the centrifugation takes place forcing the elution buffer through the membrane
(The elution buffer removes the viral RNA from this spin column)
- The viral RNA is now obtained which is free from other contaminants
Preparation of the reaction mixture for PCR amplification
- Here a master mix is used which is a premixed concentrated solution that consists of buffer, the reverse transcriptase, nucleotides (dNTPs), forward primer, the reverse primer, TaqMan probe and the DNA polymerase
- The extracted RNA template is added to the master mix by vortexing
- Then the mixture is loaded into the PCR plate which has various wells which allow analyzing of various samples at the same time
- The plate is now placed in the PCR machine which will amplify the sample
- This real-time PCR is used for the detection of new coronavirus by the amplification of target sequences in the RdRP gene, E gene and N gene
(The choice of a gene depends upon the primers and the probe sequences)
Steps of RT PCR
- Reverse Transcription
- Real-time PCR Reactions
- The PCR contains a reverse primer to which the DNA complementary strands is primed. This is now hybridized to their complementary part of the virus RNA genome
- Reverse transcriptase then adds DNA nucleotides (dNTPs) onto 3’ end of the synthesizing DNA complementary of the viral RNA
2.Real time PCR Reactions
- Initial denaturation-It leads to the denaturation of RNA-DNA hybrids. This is required for the activation of DNA polymerase and also the inactivation of reverse transcriptase
- Denaturation- It consists of heating the reaction chamber at 95° Celsius and it is used for the denaturation of double-stranded DNA template
- Annealing- In this step, the reaction temperature is lowered to 58° Celsius allowing annealing to forward primer to its complementary part of the single-stranded DNA template
- Extension- The DNA polymerase now synthesizes new DNA strands complementary to their DNA template strand by adding free nucleotides from the reaction mixture(dNTPs) that are complementary to the template in the 5’- 3’ direction
After the first cycle, the double-stranded DNA target is obtained. Then again cycle two is performed where the same denaturation annealing and extension steps are repeated
- 1st the denaturation of double-stranded DNA occurred yielding two single-stranded DNA molecule
- The reaction temperature is lowered for annealing of primers to each single-stranded DNA templates and also the TaqMan polymerase probe is added to its complementary part of the target DNA
- TaqMan polymerase consists of a fluorophore covalently attached to the 5’ end of the oligonucleotides probe
- The fluorescence is emitted by the fluorophore which is excited by cycles light source
- Also, this consists of a quencher at the emission 3’ end
- Finally in the extension DNA polymerase synthesizes new strands. When the DNA polymer reaches the TaqMan probe, its endogenous 5’ nuclease activity cleaves the probe separating that dye from its quencher
With more number of cycles, more probes are cleaved increasing the intensity of the fluorescence which also confirms the amount of amplification
The fluorophore signals can be detected by CCD cameras and can be observed in computers showing positive results of Corona.