Less than a decade after the last human outbreak caused by a zoonotic coronavirus, the Middle East respiratory syndrome (MERS) in 2012, a novel coronavirus spillover had emerged in China in 2019 and now we are witnessing a deadly pandemic. A year later, India is now facing the second wave of covid 19, the nation is struggling hard to breathe out. Widespread testing for the SARS-CoV-2 virus is important to both slow the velocity of the virus and gain information about how widespread it is.
According to the regulatory authorities such as the FDA, Food & Agriculture Organization of the United Nations (FAO), RT-PCR is one of the most accurate laboratory testing methods for the current coronavirus pandemic.
The real-time Reverse transcription-polymerase Chain Reaction (RT-PCR) test is primarily based on PCR (Polymerase Chain Reaction), a process that repeatedly copies and amplifies the specific genetic fragments of the virus, ensuring that there is enough of a sample to conduct the analysis.
Polymerase Chain Reaction (PCR) Working Principle
The principles behind every PCR are the same. Five cores ‘ingredients’ are required to set up a PCR, which are –
- The DNA template to be copied.
- Primers, short stretches of DNA that initiate the PCR reaction, designed to bind to either side of the section of DNA you want to copy.
- DNA nucleotide bases(also known as dNTPs). DNA bases (A, C, G, and T) are the building blocks of DNA and are needed to construct the new strand of DNA.
- Taq polymerase enzyme to add in the new DNA bases.
- Buffer to ensure the right conditions for the reaction.
How does the RT-PCR test work?
PCR involves a process of heating and cooling called thermal cycling which is carried out by a PCR machine.
- There are three main stages:
- Denaturing– when the double-stranded template DNA is heated to separate it into two single strands.
- Extending – when the temperature is lowered to enable the DNA primers to attach to the template DNA.
- Annealing – when the temperature is raised, and the new strand of DNA is made by the Taq polymerase enzyme
These three stages are repeated 20-40 times, doubling the number of DNA copies each time. So by the end of the reaction, the size of the new strand becomes 220-240 of the initial strand. Reverse transcription PCR, or RT-PCR, allows the use of RNA as a template. An additional step allows the detection and amplification of RNA. The RNA is reverse transcribed into complementary DNA (cDNA), using reverse transcriptase. The quality and purity of the RNA template are essential for the success of RT-PCR. The first step of RT-PCR is the synthesis of a DNA/RNA hybrid. The single-stranded DNA molecule is then completed by the DNA-dependent DNA polymerase activity of the reverse transcriptase into cDNA. The efficiency of the first-strand reaction can affect the amplification process. From here on, the standard PCR procedure is used to amplify the cDNA. The possibility to revert RNA into cDNA by RT-PCR has many advantages. RNA is single-stranded and very unstable, which makes it difficult to work with.
The PCR machine in which the Polymerase Chain Reaction takes place is known as a Thermal cycler. It works with all the necessary chemicals. During the process, one of the processing chemicals produces a fluorescent signal which only gets activated above the threshold limit of amplification, i.e., only if the coronavirus is present in enough quantity in the body.
Steps Involved in RT-PCR Test
There are essentially three steps for an RT PCR test:
The RT-PCR test starts with a simple swab taken from inside a person’s throat or nose. A swab contains a soft tip on a long, flexible stick that is inserted into the nose of the person, who is to be tested. There are different types of nose swabs including nasal swabs that collect a sample immediately inside your nostrils and nasopharyngeal swabs that go further into the nasal cavity for collection. Either type of swab is sufficient for collecting material for the COVID-19 PCR test. After collection, the swab is sealed in a tube and then sent to a laboratory.
When a laboratory technologist receives the sample, they perform a process called extraction, which isolates genetic material from the sample including genetic material from any virus that may be present. Coronaviruses have RNA or ribonucleic acid as their genetic material. However, swabs from patients yield only a tiny quantity of RNA, which is not adequate for the testing process.
To overcome this problem, the RNA — a single-strand molecule — is converted into double-stranded DNA using an enzyme. This is known as reverse transcription.
Step-3 Polymerase chain reaction
Researchers select specific areas in the genome that do not mutate rapidly as the virus evolves and create copies of these using the PCR process.
Interpreting RT- PCR test results
A positive test result means that it is very likely that the person has COVID-19. Most people have mild illnesses and can recover safely at home without medical care. It is advised to contact the healthcare providers if the symptoms get worse or if any questions or concerns arise.
A negative test result means the person probably did not have COVID-19 at the time he/she took the test. However, it is possible to be infected with SARS-CoV-2 but not have enough virus in the body to be detected by the test. For example, this may happen if a person recently became infected, but does not have symptoms, yet; or it could happen if one has had COVID-19 for more than a week before being tested.
Ups and Downs of COVID-19 PCR test
The main advantages of the COVID-19 PCR test are its accuracy and reliability. It is the most accurate test available for COVID-19 detection.
But it also has its downsides. Because the test can detect exceedingly small amounts of virus material, it can continue to detect fragments of the SARS-CoV-2 virus even after you have recovered from COVID-19 and are no longer contagious. So, people may continue to test positive even if they have had COVID-19 in the distant past, even though they cannot spread the SARS-CoV-2 virus to others.
Comparison with Antigen Test
- PCR test: This tests for the presence of the actual virus’s genetic material or its fragments as it breaks down. This is the most reliable and accurate test for detecting active infection.
- Antigen test: This test detects bits of proteins on the surface of the virus called antigens. Antigen tests are typically considered rapid, taking only 15 to 30 minutes but are less accurate than a PCR test. Rapid antigen tests are most accurate when used within a few days of the start of your symptoms, which is when the largest amount of virus is present in your body. Because this test is not as accurate as a PCR test, if an antigen test is negative, the healthcare provider may order a PCR test to confirm the negative test result.
The whole world is still struggling hard in the pandemic. The RT-PCR test plays a keys role to monitor the current situation. The accuracy of test results is really important in the first-line defense. RT-PCR is currently the most accurate test which is used in the covid 19 diagnosis tests, but like any other tests, it is also not 100% accurate. The minimum sensitivity (ability to detect positives) demanded by the Indian Council of Medical Research (ICMR) for validating an RT-PCR test is 95%. That means up to 5% false-negative results are expected. Experts recommend Covid-19 treatment for everyone showing classic symptoms irrespective of RT-PCR results.
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