Many of you may have encountered either antigen/rapid test or RT-PCR test due to the pandemic covid-19 caused by the SARS-CoV-2 virus.
Ever wondered how these tests work?
In both cases the test is done using an upper respiratory specimen which is collected in different ways; one is the most commonly used nasopharyngeal/oropharyngeal swab, in which a special swab is used to take a sample from the nose or throat. Another one is the Nasal aspirate – a saline solution will be injected into the nose and, then a sample is taken with light suction.
Antigen/rapid test
It’ is a quick and relatively costless testing method for covid-19 which can be performed without any special laboratory equipment or intensive training. Being less sensitive, it is known to give false-negative results.
Antigen test detects the presence of certain antigens, any substance that induces the immune system to produce antibodies against it, in this case, surface proteins or nucleocapsid proteins of SARS-CoV-2. The specimen collected is treated with a solution, a mixture of various reagents to break down and expose viral nucleoproteins. Here, Lateral flow immunoassay (LFIA) or Lateral flow chromatographic immunoassay is used. An LFIA is a diagnostic device used to detect the presence of certain pathogens, contaminants, or any target substance. The pregnancy test kit is one of the most commonly known lateral flow rapid test strips.
Here, the strip contains a sample pad, a conjugation pad, test line, control line, and an absorption pad- left to right. The treated sample is transferred to a slot in the testing strip. Particles in the sample are transported across the strip by capillary action. In the initial section of the strip(conjugation pad), nucleocapsid proteins or antigens get tagged with fluorescently labeled antibodies. There are two distinct lines in the testing strip, the test line, and the control line,
The test line is covered with immobile anti-SARS-CoV-2 antibodies which will stop the lateral movement of nucleocapsid proteins if there is present and the line will turn positive. The Control line indicates the test is valid; it contains labelled antibodies that bind with non-antigen targets (antigens normally found in the microbiomes of the pharynx) present in the sample which turns the control line positive. The test is read positive only if both the test line and control line shows positive. The test result is negative if only the control line is turned positive. In other cases such as no lines or only the test line appears, the test is considered to be invalid.
Real Time- Polymerase Chain Reaction (RT-PCR) test
RT-PCR test is a molecular testing method which detects the viral genetic material, specifically RNA of SARS-CoV-2. It is considered the Gold Standard for diagnosing Covid-19. All the RNA from the test specimen is collected using various reagents and methods. To make the genetic material PCR compatible, RNA is converted into cDNA (complementary DNA) molecules by using a reverse transcriptase enzyme. This collection of complementary DNAs of RNAs in the sample contains also other bacterial, viral genetic material and also human RNAs. These cDNAs undergo multiple cycles of polymerase chain reaction which involves 3 steps, denaturation-annealing-elongation.
Here during the annealing process primers used are specific to the SARS-CoV-2 virus. During the process, fluorescent probes are added to the cDNA and over cycles the number of cDNAs increases by powers 2. The increase in viral cDNA is followed by tracking the increase in fluorescence vs the number of cycles. When the level of fluorescence is above a certain threshold we can be confident that the signal is significantly above the background level.
Ct or cycle threshold is the number of cycles required to cross the threshold value of fluorescence. The lower the ct value the more RNA molecules are present in the sample, which indicates a higher viral load. Ct<40 is considered as positive for SARS-CoV-2.
Antibody tests are also used for detecting the presence of SARS-CoV-2 by testing blood sample using lateral immunoassay tests which identifies certain antibodies formed due to the infection.
After the first wave of pandemic, many governments started emphasizing on RT-PCR tests to avoid unwanted false-positive cases. We hope now you know how your swabs are used to decide whether to let you roam around or not.
References:
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