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How is Real Time RT-PCR Used in Detecting Covid 19?

Sushil Singh | 20 September, 2021

            rt-pcr test kit at home

Countries employ real time reverse transcription–polymerase chain reaction (real time RT–PCR), one of the most accurate laboratory methods for detecting, tracking, and researching the COVID-19 corona virus, as the corona virus that causes the COVID-19 disease spreads over the world.
What is a real time RT–PCR?

Real-time Reverse Transcription polymerase chain reaction defined as nuclear-based approach for identifying the presence of specific genetic material in any disease, including viruses. Originally, radioactive isotope markers were employed to detect specific genetic elements, but later refinement has resulted in the employment of unique markers, most often fluorescent dyes, to replace isotopic labeling. Unlike traditional RT–PCR, which only provides data at the conclusion of the process, this technology allows scientists to examine the results practically instantly while the procedure is still running.

Why should you go for real time RT–PCR?

The real-time RT–PCR technology is highly receptive and precise. It can provide a solid diagnosis within 3 hours though labs often take 6-8 hours. Reason being the entire procedure can be carried out within a closed tube, real time RT–PCR is substantially quicker and has a smaller risk for contagion or errors than other existing virus isolation methods. It is the most accurate and widely accepted method of detection.

How does real time RT-PCR detect COVID-19 virus?

A sample is taken from the body parts where the COVID-19 virus congregates, such as the nose or throat. Several chemical solutions are used to eliminate components such as proteins and lipids from the sample, leaving only the RNA to be extracted. This RNA is made up of the person's genetic material as well as the virus's RNA, if any is present.

A particular enzyme is used to reverse transcribe RNA to DNA. After that, scientists add small DNA pieces that are complementary to certain regions of the transcribed viral DNA. These fragments attach themselves to target portions of viral DNA if the virus is present in the sample. These fragments attach themselves to target portions of viral DNA if the virus is present in the sample. Some of the genetic fragments added during amplification are used to form DNA strands, while others are used to synthesise DNA and add flag labels to the strands, which are subsequently used to detect the virus.

After that, the mixture is put into an RT–PCR machine. The machine alternates between heating and cooling the mixture, triggering chemical reactions that create fresh, identical copies of the viral DNA segments of interest. Every cycle doubles the number before it: two copies become four, four copies become eight, and so on. A typical real-time RT–PCR setup goes through 35 cycles, which means that each strand of virus in the sample generates roughly 35 billion new copies of viral DNA by the end of the procedure.

The marker labels connect to the DNA strands as new copies of the viral DNA sections are made, releasing a fluorescent dye that is monitored by the machine's computer and displayed in real time on the screen. After each cycle, the computer records the quantity of fluorescence in the sample. When a certain degree of fluorescence is reached, it confirms that the organism is alive.
The cycles it takes are recorded by the scientists to keep a track record of how viral is the infection. The fewer the cycles are, the more serious the infection.
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