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 markers to detect targeted genetic materials, but subsequent refining has led to the replacement of isotopic labeling with special markers, most frequently fluorescent dyes. This technique allows scientists to see the results almost immediately while the process is still ongoing, 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 for diagnosing other diseases, such as the Ebola virus and Zika virus, many need support in adapting this method for the COVID-19 virus, as well as in increasing their national testing capacities.
How does real-time RT–PCR work with the COVID-19 virus?
A sample is collected from the parts of the body where the COVID-19 virus 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 that extract only the RNA present in the sample. This extracted RNA is a mix of the person’s own genetic material and, if present, the virus’s 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. If the virus is present in a sample, these fragments attach themselves to target sections of the viral DNA. Some of the added genetic fragments are used for building DNA strands during amplification, while the others are used 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 an 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 is repeated over and over to continue 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 set-up 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 a certain level of fluorescence is surpassed, 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 next-day after the test is initialized. 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 carried out within a closed tube. It continues to be 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 for understanding the development and spread of the virus, 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 which may have developed and spread without symptoms.