From UPSC perspective, the following things are important :
Prelims level : Polymerase Chain Reaction Test
Mains level : Coronovirus outbreak and its mitigation
The diagnosis of COVID-19 can be done with the Polymerase Chain Reaction (PCR) Test which is explained as under:
The PCR Test
- It uses a technique that creates copies of a segment of DNA. ‘Polymerase’ refers to the enzymes that make the copies of DNA.
- Kary Mullis, the American biochemist who invented the PCR technique, was awarded the Nobel Prize for Chemistry in 1993.
- The ‘chain reaction’ is how the DNA fragments are copied, exponentially — one is copied into two, the two are copied into four, and so on.
- However, SARS-COV-2 is a virus made of RNA, which needs to be converted into DNA. For this, the technique includes a process called reverse transcription.
- A ‘reverse transcriptase’ enzyme converts the RNA into DNA. Copies of the DNA are then made and amplified.
- A fluorescent DNA binding dye called the “probe” shows the presence of the virus. The test also distinguishes SARS-COV-2 from other viruses.
1) Collection and transport
- Testing centre takes swabs from nasal cavities and back of the throat (pharynx), and puts samples in a “virus transport medium”, which contains balanced salts and albumin to prevent the virus from disintegrating.
- Sample is then transported in cold storage to the testing lab.
2) Extraction of viral RNA
- Coronaviruses have large single-stranded RNA genomes.
- Testing lab extracts the RNA from the samples, using commercially available kits.
3) Putting THE RNA in THE PCR mix
- Extracted RNA is added to a polymerase chain reaction (PCR) mix.
- This includes the ‘master mix’, which contains a ‘reverse transcriptase’ enzyme that converts the RNA into DNA.
- Master Mix contains Taq polymerase, the enzyme that creates copies of the DNA, nucleotides, as well as other elements such as magnesium — an ion of which is needed to amplify the DNA.
- The PCR mix also contains ‘reagents’ such as ‘primers’ and ‘probes’.
- Primers are particular strands of DNA that are designed to bind with the DNA that is to be copied; probes are used to detect the specific sequence in the DNA sample.
- Finally, the PCR mix consists of a “housekeeping” gene — a normal human gene (RNAse P) that is used to ensure that samples were properly collected, and RNA extracted.
4) Amplification of the viral DNA
- Sample, in its PCR mix, is put into tubes or plates, which are then put in a thermal cycler machine that is used to conduct the PCR process.
- First, the RNA is converted into DNA. Then the process of copying the genes starts.
- The thermal cycler heats and cools the mixture with the sample, alternating between three temperatures — for melting the DNA to separate the two strands.
- The thermal cycler runs 30-40 such cycles in order to amplify the DNA to check for the virus.
5) Testing against controls
- Amplified DNA is tested against a positive control, which usually consists of genes of the virus cloned into plasmid, and a negative control, which is a ‘known’ sample that has tested negative for the virus earlier.
- RNase P should show amplification, positive control should be positive, negative control should be negative, and then whatever result you get for the specimen, is the correct result.
- In order for a test to be valid before the result is released, certain ‘validity criteria’ have to be met.
- If the housekeeping gene (RNase P) is positive, positive control is positive, negative control is negative, and the sample does not show any PCR positive result, the sample is declared negative.
- If the PCR result is positive, the patient has COVID-19.