As the coronavirus that causes COVID-19 spreads around the world, the IAEA, in partnership with the Food and Agriculture Organization of the United Nations (FAO), offers its support and expertise to help people countries to use real-time reverse transcription-polymerase. chain reaction (real-time RT-PCR), one of the fastest and most accurate laboratory methods for detecting, tracking, and studying the COVID-19 virus.
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 updated details on viruses and genetics.
What is real-time RT-PCR?
Real-time RT-PCR is a nuclear-based method to detect the presence of specific genetic material in any pathogen, including a virus. Originally, the method used radioactive isotope markers to detect specific genetic materials, but subsequent refinement has led to the replacement of the isotopic labelling by special markers, most often fluorescent dyes. This technique allows scientists to see the 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 the Zika virus, many need support to adopt this method for the COVID-19 virus, as well as to increase their national testing capabilities.
What is a virus? What is genetic material?
A virus is a microscopic bundle of genetic material surrounded by a molecular envelope. This genetic material can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
DNA is a two-stranded molecule found in all organisms, including animals, plants, and viruses, and contains the genetic code, or blueprint, of how these organisms are made and developed.
RNA is generally a single-stranded molecule that copies transcribes, and transmits parts of the genetic code to proteins so that they can synthesize and perform functions that keep organisms alive and developing. Different variations of RNA are responsible for copying, transcribing and transmitting.
Some viruses, such as the SARS-CoV-2 coronavirus, which causes COVID-19, only contain RNA, which means they depend on the infiltration of 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 control and “reprogram” the cells, turning them into virus production factories.
For a virus-like COVID-19 to be detected early in the body using real-time RT-PCR, scientists must convert 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 virus detection.
Scientists amplify a specific part of the viral DNA transcribed hundreds of thousands of times. Amplification is important so that, rather than trying to detect a minuscule amount of the virus among millions of strands of genetic information, scientists have a large enough number of the target sections of viral DNA to accurately confirm that the virus is present.
How does real-time RT-PCR work with the COVID-19 virus?
A sample is collected from parts of the body where the COVID-19 virus accumulates, 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 that extract only the RNA present in the sample. This extracted RNA is a mixture of the person’s own genetic material and, if present, the RNA of the virus.
RNA is reverse transcribed to DNA using a specific enzyme. The scientists then add additional short pieces of DNA that are complementary to specific parts of the transcribed viral DNA. If the virus is present in a sample, these fragments adhere to target sections of the viral DNA. Some of the added gene fragments are used to build DNA strands during amplification, while others are used to build DNA and add marker tags to the strands, which are then used to detect the virus.
Then the mixture is placed in an RT-PCR machine. The machine goes through temperatures that heat and cool the mixture to trigger specific chemical reactions that create new and identical copies of the target sections of the viral DNA. The cycle is repeated over and over again to continue copying the target sections of the 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 the viral DNA sections are built, the marker labels 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. . The computer tracks the amount of fluorescence in the sample after each cycle. When a certain level of fluorescence is exceeded, it is confirmed that the virus is present. Scientists also monitor how many cycles it takes to reach this level to estimate the severity of the infection: the fewer cycles, the more severe the viral infection.
Why use real-time RT-PCR?
The real-time RT-PCR technique is highly sensitive and specific and can provide a reliable diagnosis in as little as three hours, although labs take an average of six to eight hours. Compared to other available virus isolation methods, real-time RT-PCR is significantly faster and has less potential for contamination or errors, as the entire process can be carried out within a closed tube. It remains the most accurate method available for the detection of the COVID-19 virus.
However, real-time RT-PCR cannot be used to detect past infections, which is important to understand virus development and spread, since viruses are only present in the body for a specific period of time. Other methods are needed to detect, track, and study past infections, particularly 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 variation of PCR or polymerase chain reaction. The two techniques use the same process, except that RT-PCR has an additional RNA-to-DNA reverse transcription step, or RT, to allow for 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 contain RNA that needs to be transcribed into DNA for amplification. Both techniques can be performed in “real-time”, which means that the results are visible almost immediately, 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, African swine fever, and foot-and-mouth disease. Since the COVID-19 virus contains only RNA, real-time or conventional RT-PCR is used to detect it.