Explain the working of RT-PCR tests

Real-Time RT-PCR or fully known as Reverse Transcription Polymerase Chain Reaction is a sensitive and fast test that is done for detecting the presence and existence of exact genetic materials within a sample. This genetic material can be definite to bacteria, humans, and viruses like SARS-CoV-2.

The base of Real-Time RT-PCR is derived from Polymerase Chain Reaction (PCR); which is a laboratory technique that was developed by Nobel Prize-winner, Kary B. Mullis, in the 1980s. It allowed the researchers to intensify and detect specific DNA targets (1, 2). Further, this technology was improved to allow real-time quantification and visualization of DNA targets as they go through amplification. In order to visualize the whole amplification of DNA, RTPCR uses the increase in fluorescence intensity of a fluorogenic probe in proportion to the amount of amplified DNA.

How does RTPCR work?

Test Collection: To begin the symptomatic test, a trained healthcare person will utilize a swab to gather nasopharyngeal examples from the patient's nasopharynx. The sample is then positioned into a sterile cylinder tube containing viral transport media to keep the virus feasible.

Test Preparation: Once the examples show up at the lab, specialists will utilize commercial purification kits to remove RNA from the sample. Then, the RNA test is added into one reaction mixture containing every one of the ingredients expected to finish the test, otherwise called "one-step RT-PCR".

Reverse Transcription: As mentioned prior, PCR only works on DNA samples. Hence, the role of reverse transcriptase inside the response blend is to change over all the RNA present inside a given example into cDNA. This incorporates human RNA, bacterial RNA, other Covid RNA and if present, SARS-CoV-2's RNA.

Stage 1 - Separation: To start, it is vital to recall that DNA is a double-stranded structure. Along these lines, the subsequent stage is to loosen up the DNA atom into discrete DNA stands. This is achieved by warming the DNA to high temperatures (> 90°C) for a sum of 10 min.

Stage 2 - Primer Annealing: Next, is the addition of short pieces of DNA, called primers. Primers are planned with high explicitness and will connect to explicit focuses inside the DNA of the SARS-CoV-2 RNA infection. A particularly low temperature is required for preliminary toughening as well. As a general rule, there 7 normal quality targets utilized for testing COVID-19; every quality objective is vital for the infection's replication or structure (4).

Stage 3 - Primer Extension/Elongation: Since DNA is a double-stranded structure, there are two primers in this mixture. When the preliminaries connect to their objective DNAs, they will guide the DNA polymerase on where to start and complete intensification on the DNA portion. This progression brings about an indistinguishable DNA duplicate of the objective DNA.

Also, Real-Time PCR will repeat the cycle on various occasions (for about 40 cycles). Each time RT-PCR finishes a cycle, it will double the target DNA. Moreover, there are additionally fluorescent tests that tight spot explicitly to the DNA targets, downstream of every groundwork.

Since the cases of coronavirus in Delhi are on the rise, the RTPCR is a very important document. If you are travelling domestic, there is no requirement but if travelling internationally you will be asked to present the negative RTPCR report. Only then will you be permitted to fly.