Welcome to fosterbrain.com dear visitors, you all might be aware of the test which detects the presence of Corona virus. Covid-19 is detected by a test called Real time PCR. Reading this article will make you understand what is PCR and also you will be able to learn about the detection process of Covid 19.
Now, you must be wondering what's the full form of RT-PCR?
It's called Real time Reverse Transcription Polymerase Chain Reaction.
To know it in details firstly you must understand what is a Polymerase Chain Reaction. PCR was invented by Kary Mullis in 1984. It is a method by which we can make millions to billions copies of our DNA sample.
Now what is DNA? It stands for Deoxyribo Nucleic Acid. It is our genetic material. It holds all the instructions for our growth and development.
Like a guide book DNA contains all the information for making all the proteins required for our bodies. It is a double helical structure which means it has two strands arranged in a helix.
Helix means a twisted spiral shape. Its structure resembles a twisted ladder. It is made up of building blocks called nucleotides.
Nucleotides are made of three parts:
A phosphate group, a five carbon sugar group called deoxyribose because it lacks an oxygen group on one of its carbons and lastly one of the four types of nitrogen bases.
Nitrogen base is an organic molecule with a nitrogen atom which has the properties of a base.
There are mainly 4 nitrogenous bases:
- Adenine (A)
- Cytosine (C)
- Guanine (G) and
- Thymine (T)
The sequence of these bases form the instructions. Each strand of DNA is composed of long sequences of these four bases, A, C, G and T linked into chains with alternating phosphate and sugar groups.
The bases always pair in the same way, A pairs with T, C with G. They are joined together by hydrogen bonds. The bases on one strand of the DNA pairs with the complementary bases on the opposite strand of DNA.
Each strand of DNA has a beginning called 5’ and an end called 3’. The two strands run in the opposite direction to each other which means one runs from 5’ to 3’ and the other runs from 3’ to 5’.
So they are also called antiparallel. This structure of DNA was discovered by Francis Crick and James Watson.
For almost all of the living beings DNA is their hereditary material.
But there exists some viruses which are called retroviruses whose genetic material is RNA. It stands for Ribo Nucleic Acid.
RNA is single stranded unlike the DNA and it consists of nitrogenous bases which are almost same as DNA, they are Adenine, Guanine, Cytosine but in place of Thymine it contains Uracil.
And the sugar group in the RNA is a ribose sugar which means it doesn't lack an oxygen group which lacks in DNA.
Now let's come to our main topic i.e PCR
How is PCR done?
The machine in which PCR is performed is simply called a Thermocycler.
The DNA mixture are poured in test tubes and are put into the machine, it changes the temperature of the mixture so that it suits each step of the process.
What are the components of the DNA mixture?
- the DNA segment: the target DNA sequence which we want to make copies
- specific primers: short single stranded DNA sequences
- heat resistant enzyme: mostly an enzyme called Taq Polymerase
- the four different types of DNA nucleotides: A,T,G,C
- the salts needed for the enzyme to act.
So, PCR generally involves 3 steps
Denaturation- In this step, the two stands of the DNA double helix are separated.
This happens by raising the mixture's temperature, which causes the hydrogen bonds between the complementary DNA to break. The temperature in this step is 94–98 °C .
Annealing- The temperature of the solution is lowered in this step due to which the primers bind to the DNA. One primer binds with each strand. Here the temperature is 50–65 °C.
Extension- Using the original DNA strands as templates, new strands are made. An enzyme called DNA Polymerase joins the free DNA nucleotides together.
The order in which the nucleotides are added is determined by the sequence of nucleotides in the original DNA strand. The temperature is maintained at 75–80 °C in this step.
After these steps, one cycle of PCR is completed resulting in two double stranded sequences of the DNA, each containing one new strand and one original strand.
The cycle when repeated many times can produce million copies of DNA. It takes 2–3 hours to get almost billion copies of DNA.
That was all about the general process of PCR. PCR technology is developing day by day. Various types of PCR are developed till date. Real time PCR is one such type.
RT-PCR is a technique where an enzyme called Reverse Transciptase (RT) is used to generate complementary DNA from an RNA template.
This process of generating complementary DNA is called reverse transcription. RT-PCR is generally used to measure the amount of a specific RNA which is done by monitoring the reaction using florescent dyes.
The use of florescent dyes allows us to see the results immediately during the process so it is called real time RT-PCR.
So now, before discussing about the process of the RT-PCR test for Covid-19, let us know something about the virus which causes the disease.
Which virus is responsible for Covid-19?
The virus strain which causes the Coronavirus disease 2019 is the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
It is a single stranded RNA virus which means its genetic material is RNA and hence it is a retrovirus.
This virus spreads among people through close contact and through respiratory droplets produced when an infected person sneezes or coughs.
This virus binds with the respiratory cells and uses them to produce more viruses.
How RT-PCR test is done to detect Covid-19?
A sample is collected from the suspected person’s nose or throat using a swab. Swab means a small piece of soft material used for taking a small amount of substance from a body.
The sample is then treated with several chemicals that extract only the RNA present in the sample and removes other substances such as proteins and fats.
This extracted RNA is a mix of the person’s own genetic material and the virus’s RNA, if present.
The RNA is then reverse transcribed to DNA. Then they add additional short segments of DNA that are complementary to specific parts of the transcribed viral DNA.
If the virus is present then these segments attach themselves to target viral DNA.
The mixture is then placed in an RT–PCR machine that creates new, identical copies of the target sections of viral DNA.
The cycle is repeated again and again to copy the target sections of viral DNA. After each cycle the number doubles: two copies become four, four copies become eight, and so on.
A standard real time RT–PCR set-up generally performs 35 cycles, which means at the end around 35 billion new copies are created of the sections of viral DNA from each strand of the virus in the sample.
As the new copies of the viral DNA are made, the marker attaches to the DNA strands and then releases a fluorescent dye, which is measured by the computer and is presented on the screen in real time.
After each cycle it tracks the amount of fluorescence in the sample. When a certain level of fluorescence is crossed then it confirms that the virus is present.
It is also measured that how many cycles it takes to reach this level so that the severity of the infection can be estimated, less number of cycles means that the infection is more severe.
So that was all about the RT-PCR test used to detect Covid-19, I have tried to explain it in a very simple way. Hope you all will like it.
If you want me to explain anything more let me know in the comment section.
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