Dr V Ravi, Senior Professor and Head, Department of Neurovirology, NIMHANS, explains the process of detecting the coronavirus to The Indian Express. Here are the five steps:
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.
Extraction of viral RNA
Coronaviruses such as the SARS-CoV (which emerged in China in 2002-03), MERS-CoV (which appeared in Saudi Arabia in 2012), or the current SARS-CoV-2 which has caused the COVID-19 pandemic, have large, single-stranded RNA genomes. Testing lab extracts the RNA from the samples, using commercially available kits (such as those made by companies like QIAGEN.)
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. WHO has recommended specific primers and probes for testing for COVID-19.
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.
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, for the primer to bind to the DNA, and for synthesising a new strand — all within one cycle that lasts a minute. The thermal cycler runs 30-40 such cycles in order to amplify the DNA to check for the virus.
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,” said neurovirologist Dr V Ravi. 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 for the SARS-CoV-2 virus RNA. If the PCR result is positive, the patient has COVID-19.