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How is the COVID-19 Virus Detected using Real Time RT-PCR?

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One of the most widely used and accurate laboratory methods for detecting the novel coronavirus is real time RT-PCR. (Photo: IAEA)

As the virus that causes the COVID-19 disease spreads across 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 use real time reverse transcription–polymerase chain reaction (real time RT-PCR), one of the most accurate laboratory methods for detecting, tracking, and studying the coronavirus.

But what is real time RT-PCR? How does it work?  And what does it have to do with nuclear technology? Here’s a handy overview of the technique, how it works and a few refresher 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 from any pathogen, including a virus. Originally, the method used radioactive isotope markers to detect targeted genetic materials, but subsequent refining has led to the replacement of the isotopic labelling with special markers, most frequently fluorescent dyes. With this technique, scientists can see the results almost immediately while the process is still ongoing; conventional RT-PCR only provides results at the end.

While real time RT-PCR is now the most widely used method for detecting coronaviruses, many countries still need support in setting up and using the technique.

What is a virus? What is genetic material?

A virus is a microscopic package of genetic material surrounded by a molecular envelope. The genetic material can be either DNA or RNA. 

DNA is a two-strand molecule that is found in all organisms, such as animals, plants, and viruses, and it holds the genetic code, or blueprint, for how these organisms are made and develop.

RNA is generally a one-strand molecule that copies, transcribes and transmits parts of the genetic code to proteins so they can synthetize and carry out functions that keep organisms alive and developing. There are different variations of RNA that do the copying, transcribing and transmitting.

Some viruses such as the coronavirus (SARS-Cov2) only contain RNA, which means they rely on infiltrating healthy cells to multiply and survive. Once inside the cell, the virus uses its own genetic code — RNA in the case of the coronavirus — to take control of and ‘reprogramme’ the cells so that they become virus-making factories.  

In order for a virus like the coronavirus to be detected early in the body using real time RT-PCR, scientists need to convert the RNA to DNA. This is a process called ‘reverse transcription’. They do this because only DNA can be copied — or amplified — which is a key part of the real time RT-PCR process for detecting viruses.

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

How does real time RT-PCR work with the coronavirus?

A sample is collected from parts of the body where the coronavirus gathers, such as a person’s nose or throat. The sample is treated with several chemical solutions that remove substances, such as proteins and fats, and extracts only the RNA present in the sample. This extracted RNA is a mix of a person’s own genetic material and, if present, the coronavirus’ RNA.

The RNA is reverse transcribed to DNA using a specific enzyme. Scientists then add additional short fragments of DNA that are complementary to specific parts of the transcribed viral DNA. These fragments attach themselves to target sections of the viral DNA if the virus is present in a sample. Some of the added genetic fragments are for building DNA strands during amplification, while the others are for building the DNA and adding marker labels to the strands, which are then used to detect the virus.

The mixture is then placed in a RT-PCR machine. The machine cycles through temperatures 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 to continue copying the target sections of viral DNA. Each cycle doubles the previous amount: two copies become four, four copies become eight, and so on. A standard real time RT-PCR setup usually goes through 35 cycles, which means that by the end of the process, around 35 billion new copies of the sections of viral DNA are created from each strand of the virus present in the sample.

As new copies of the viral DNA sections are built, the marker labels attach to the DNA strands and then release a fluorescent dye, which is measured by the machine’s computer and presented in real time on the screen. The computer tracks the amount of fluorescence in the sample after each cycle. When the amount goes over a certain level of fluorescence, this confirms that the virus is present. Scientists also monitor how many cycles it takes to reach this level in order to estimate the severity of the infection: the fewer the cycles, the more severe the viral infection is.

Why use real time RT-PCR?

The real time RT-PCR technique is highly sensitive and specific and can deliver a reliable diagnosis as fast as three hours, though usually laboratories take on average between 6 to 8 hours. Compared to other available virus isolation methods, real time RT-PCR is significantly faster and has a lower potential for contamination or errors as the entire process can be done within a closed tube. It continues to be the most accurate method available for detection of the coronavirus.

To detect past infections, which is important for understanding the development and spread of the virus, real time RT-PCR cannot be used as viruses are only present in the body for a specific window of time. Other methods are necessary to detect, track and study past infections, particularly those that may have developed and spread without symptoms.

The IAEA, in partnership with the FAO, has trained and equipped experts from all over the world to use the real time RT-PCR method for over 20 years particularly through its VETLAB network of veterinary diagnostic laboratories. Recently, this technique has also been employed to diagnose other diseases such as Ebola, Zika, MERS-Cov, SARS-Cov1, and other major zoonotic and animal diseases. Zoonotic diseases are animal diseases that can also infect humans. 

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