RT-PCR, antigen, antibody, TrueNAT — all you need to know about the different Covid tests

Each kind of test has different processes, prices & also purposes — antigen tests can identify positive cases, while antibody tests can screen a wide area.

A scientist performing an RT-PCR test | Photo: Angana Chakrabarti | ThePrint

New Delhi: India has scaled up testing for Covid-19 to over 2 lakh tests per day. But that figure doesn’t include all the types of tests available, which all serve different purposes.

The testing figures that ICMR updates every day include data only from the RT-PCR, TrueNAT and CBNAAT tests — with the latter two being tuberculosis tests roped in for Covid testing last month.

Neither antibody tests, which are for surveillance purposes, nor the newly introduced antigen tests are included in the ICMR’s data.

ThePrint explains the differences between all these types of tests.

RT-PCR

The real-time polymerase chain reaction (RT-PCR) test is the most commonly used one for Covid-19. It is primarily based on PCR, a process that repeatedly copies and amplifies the specific genetic fragments of the virus, ensuring that there is enough of a sample to conduct the analysis.

The RT-PCR test starts with a simple swab taken from inside a person’s throat or nose. Coronaviruses have RNA or ribonucleic acid as their genetic material. However, swabs from patients yield only a tiny quantity of RNA, which is not adequate for the testing process.

To overcome this problem, the RNA — a single-strand molecule — is converted into a two-strand DNA using an enzyme. This is known as reverse transcription.

Researchers select specific areas in the genome that do not mutate rapidly as the virus evolves, and create copies of these using the PCR process.

The next step is the use of primers. These are small pieces of DNA designed only to bind to the selected DNA sequence of the SARS-CoV-2’s viral genome. Along with primers, the process uses a fluorescent dye, which acts as the probe.

The patient sample, primer and probe are together left in the PCR machine for the binding process to take place. A fluorescent signal marks the presence of the virus.

The time duration for the testing process is around four to eight hours, but results may take up to a day to arrive because of the time taken to transport samples to labs.

The RT-PCR test is also the most expensive of all those used for Covid-19. Initially, the cost of the test was capped at Rs 4,500 by the ICMR. However, this cap was recently removed and states were asked to fix costs instead.

Antigen testing 

The new antigen tests that have been approved for Covid-19 diagnosis in India give results in 30 minutes.

These tests are designed to detect a specific protein in the virus that elicits the body’s immune response. In the case of Covid-19, it is the ‘spike protein’ present on the surface of the coronavirus that facilitates its entry into the human cell.

For this test, professionals collect a nasal swab, which is then immersed in a solution that deactivates the virus.

A few drops of this solution are then put on a test strip. This has to be done within an hour of the immersion of the swab in the solution.

The test strips contain artificial antibodies designed to bind to coronavirus proteins. If a person is infected with coronavirus, the test lines will appear on the paper strips within 15 minutes.

Since antigen testing does not involve any amplification process, swab samples may lack enough antigen material to be detectable. This may result in false negative tests.

For this reason, if a person tests negative through antigen testing, they still need to get an RT-PCR test done for confirmation. If a person tests positive, however, a confirmation RT-PCR is not required.

The advantage of using this test is that it reduces the burden of relying on just RT-PCR tests to identify Covid-19 patients.

“Antigen testing is useful because even if it’s less sensitive, it is rapid and the results that are positive will be positive. So, patients who test positive can get into isolation faster,” Dr Gagandeep Kang, executive director of the Transnational Health Service and Technology Institute, told ThePrint in an earlier interview.

She added that antigen tests remove about half of the positives from the testing load.

Antigen tests are much cheaper than the RT-PCR, and cost Rs 450 each.

Antibody tests 

Antibody tests, also known as serological tests, detect whether a person has antibodies to the virus.

Antibodies are naturally produced by the body’s immune system to fight off infections. Antibody tests cannot be used to diagnose Covid-19, but can reveal whether a person was recently exposed to the virus.

Persons with antibodies in their bloodstream are likely to have immunity to the disease, although scientists are still not sure how long Covid-19 antibodies offer protection from the infection.

For the antibody test, trained professionals collect a few drops of blood. The sample is placed on a cassette or cartridge that contains the SARS-CoV-2 proteins.

If the blood samples contain antibodies, they will immediately bind to the viral proteins. The positive result is indicated in the form of lines, like a home pregnancy test.

Antibody tests can be of different types. One of the tests, known as Enzyme-Linked Immunosorbent Assay (ELISA), which is designed specifically for screening large numbers of specimens at a time, is suitable for detecting the spread of the disease in a large population within a short span of time.

Antibody tests can be useful to carry out surveys to check whether a population has been exposed to the virus. These are currently only being used for research and surveillance purposes.

TrueNAT

On 19 May, the ICMR revised its testing guidelines to allow the use of the TrueNAT system for screening and confirmation of Covid-19.

The chip-based test works by detecting the SARS-CoV-2 E-gene, which helps build the envelope that holds the components of the coronavirus together, and the gene for the RNA-dependent RNA polymerase, an enzyme that enables the virus to replicate.

Unlike traditional RT-PCR tests, the sample preparation in TrueNAT tests is automated, and the results are available within half an hour.

The test uses nose or throat swab samples, which are collected from patients and dipped in a solution that inactivates the virus.

A few drops of the solution are then placed on a cartridge. On inserting this cartridge into a machine, a pre-programmed reaction is initiated, which extracts the nucleic acids or the genetic material from the samples.

This has to be followed by an RT-PCR. The purified nucleic acid is added into a micro-tube containing freeze-dried RT-PCR reagents, and the solution is allowed to stand for about a minute.

This solution is then applied to a microchip and the test is inserted into another machine, where the reverse transcription and PCR take place.

The advantage of this test is that it is quick and portable. This allows teams to set up mobile testing centres or kiosks in containment zones, instead of having to transport samples to labs.

TrueNAT is an indigenously developed, portable version of CB-NAAT or Cartridge Based Nucleic Acid Amplification Test, also known as the Genexpert test. Both these tests were initially developed for testing tuberculosis.

While both versions are cheaper and quicker than RT-PCR tests, TrueNAT is battery operated and portable, while CB-NAAT machines need an uninterrupted power supply and air conditioning, and hence cannot be deployed in containment zones.

TrueNAT testing kits are priced at Rs 1,200.