How is the COVID-19 virus detected by real-time RT-PCR?

As the coronavirus that causes the disease COVID-19 spreads around the world, the IAEA, in partnership with the Food and Agriculture Organization of the United Nations (FAO), is offering its support and expertise to help countries to use real-time reverse transcription-polymerase. chain reaction (real-time RT-PCR), is one of the most accurate laboratory methods to detect, track and study the COVID-19 coronavirus.

But what is real-time RT-PCR? How does it work? How is it different from PCR? And what does this have to do with nuclear technology? Here's a helpful overview of the technique, how it works, and some up-to-date details on viruses and genetics.

What is real-time RT-PCR?

Real-time RT-PCR is a nuclear-derived method for detecting the presence of specific genetic material in any pathogen, including a virus. Originally the method used radioactive isotope labels to detect specific genetic material, but further refinements have led to the replacement of isotope labeling with special labels, most commonly fluorescent dyes. This technique allows scientists to see results almost immediately while the process is still in progress, whereas conventional RT-PCR only provides results at the end of the process.

Real-time RT-PCR is one of the most widely used laboratory methods for detecting the COVID-19 virus. While many countries have used real-time RT-PCR to diagnose other diseases, such as the Ebola virus and Zika virus, many need support to adapt this method to the COVID-19 virus, as well as to increase their Nationals test abilities.

What is a virus? What is genetic material?

A virus is a microscopic set of genetic material surrounded by a molecular envelope. This genetic material can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

DNA is a double-stranded molecule found in all organisms, such as animals, plants, and viruses, and contains the genetic code, or blueprint, for how these organisms are created and developed.

RNA is usually a single-stranded molecule that copies, transcribes, and transmits parts of the genetic code into proteins so that they can synthesize and perform functions that keep organisms alive and developing. Different RNA variants are responsible for copying, transcription, and transmission.

Some viruses like the coronavirus (SARS-CoV-2), which causes COVID-19, contain only RNA, meaning they rely on infiltration by healthy cells to multiply and survive. Once inside the cell, the virus uses its own genetic code, RNA in the case of the COVID-19 virus, to take over and "reprogram" the cells, turning them into virus factories.

For a virus like COVID-19 to be detected early in the body using real-time RT-PCR, scientists need to convert RNA into DNA. This is a process called "reverse transcription". They do this because just DNA can be copied or amplified, which is a key element of the real-time RT-PCR process for detecting viruses.

Scientists amplify a specific part of transcribed viral DNA hundreds of thousands of times. Amplification is important so that instead of trying to detect a little amount of the virus among millions of strands of genetic information, scientists have a large enough number of target sections of viral DNA to accurately verify. that the virus is present.

How does real-time RT-PCR work with the COVID-19 virus?

A sample is taken from parts of the body where the COVID-19 virus is accumulating, such as a person's nose or throat. The sample is treated with various chemical solutions that remove substances such as proteins and fats and extract only the RNA present in the sample. This extracted RNA is a mix of the person's own genetic material and, if present, RNA from the virus.

RNA is transcribed back into DNA using a specific enzyme. Scientists then add additional short pieces of DNA that are complementary to specific parts of the transcribed viral DNA. If virus is present in a sample, these fragments adhere to target sections of viral DNA. Some of the added genetic fragments are used to build DNA strands during amplification, while others are used to build DNA and add markers to the strands, which are then used to detect the virus.

The mixture is then placed in an RT-PCR device. The machine goes through temperature cycles that heat and cool the mixture to trigger specific chemical reactions that create new, identical copies of the target sections of viral DNA. The cycle repeats over and over again to keep copying the target sections of viral DNA. Each cycle doubles the previous number: two copies become four, four copies become eight, and so on. A standard real-time RT-PCR setup typically goes through 35 cycles, which means that at the end of the process, about 35 billion new copies of viral DNA sections are created from each strand of virus present in the sample.

As new copies of sections of viral DNA are made, the markers adhere to the DNA strands and then release a fluorescent dye, which is measured by the machine's computer and displayed in real time on the screen. 'screen. A computer tracks the quantity of fluorescence in the sample after each cycle. When a certain level of fluorescence is exceeded, this confirms the presence of the virus. Scientists also monitor the number of cycles needed to reach this level to estimate the severity of infection: the fewer cycles, the more severe the viral infection.

Why use real-time RT-PCR?

The real-time RT-PCR method is highly sensitive and specific and can provide a reliable diagnosis in as short as three hours, although labs take six to eight hours on average. Compared to other virus isolation methods available, real-time RT-PCR is significantly faster and has less risk of contamination or errors because the entire process can be performed inside a closed tube. It remains the most accurate method available for detecting the COVID-19 virus.

However, real-time RT-PCR cannot be used to detect past infections, which is important for understanding the development and spread of viruses because viruses are only present in the body for a specific period of time. . Other methods are needed to detect, track, and study past infections, especially those that may have developed and spread without symptoms.

What is PCR and how is it different from real-time RT-PCR?

RT-PCR is a variant of Polymerase-Chain Reaction (PCR). Both techniques use the same process, except that RT-PCR has an additional step of RNA-DNA reverse transcription, or RT, to enable amplification. This means that PCR is used for pathogens, such as viruses, and bacteria, that already contain DNA for amplification, while RT-PCR is used for those that have RNA that needs to be amplified. be transcribed into DNA for amplification. Both methods can be performed in "real-time", which means that the results are visible almost directly, whereas when used "conventionally", the results are only visible at the end of the reaction.

PCR is one of the most widely used diagnostic tests to detect pathogens, including viruses, that cause diseases such as Ebola virus, African swine fever and foot and mouth disease. Since the COVID-19 virus contains only RNA, conventional or real-time RT-PCR is used to detect it. For more information visit our website.