What is Real Time RT-PCR?

Study adapted from IAEA

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. 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. 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.

How Does It Work?

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 We Need RT-PCR Testing?

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.

Study adapted from IAEA