We all are aware of the spread of COVID-19 across the world. As vaccination has started, many institutes have come forward to offer their support and expertise to deal with the pandemic. For example, in partnership with the Food and Agriculture Organization of the United Nations (FAO), the IAEA has been helping countries use real time PCR. This laboratory method is entirely accurate for detecting, tracking, and studying the COVID-19 coronavirus.
There are many questions that linger on about real-time PCR tests. What is real time RT–PCR? How does it work, and is it different from PCR?
This blog attempts to provide you with a handy overview of the technique, how it works and addresses many other questions.
Before we find out about RT-PCR, let us first understand what a virus is; and what is its genetic material? A virus is an invisible package of genetic material adjoined by a molecular envelope. This genetic material can be categorized into either DNA or RNA.
DNA is a 2-strand molecule found in all living beings such as plants, animals, and viruses. This means that every organism has the genetic code or blueprint of how they are made.
RNA is usually a 1-strand molecule that transmits, copies, and transcribes parts of the genetic code to proteins. It synthesizes functions that keep organisms alive and developing. Multiple variations of RNA cause transmitting, copying, and transcribing.
Some viruses like coronavirus or SARS-CoV-2 contain only the RNA. These viruses infiltrate healthy cells and multiply themselves to survive. After going inside the cell, this virus employs its own genetic code to control or reprogram the cells, transforming them into an incubator for viruses.
PCR or polymerase chain reaction tests detect this genetic signature that the virus carries.
What is RT-PCR test?
Real-time RT–PCR is a nuclear-derived technique for detecting signature genetic material of pathogens like virus or bacteria. Initially, this technique employed radioactive isotope markers to spot targeted genes. After subsequent refining, the method replaced isotopic labeling with unique markers- more specifically as fluorescent dyes.
The RT PCR technique helps scientists see the results immediately while the process is still on. In contrast, the traditional RT–PCR provide results only at the end of the process.
Many laboratories use real-time RT PCR, making it one of the most widely used methods for detecting the COVID-19 virus. Several countries have used real-time RT PCR for diagnosing other viruses or diseases like the Ebola virus and Zika virus. Many still need assistance in adapting this technique to spot the COVID-19 virus and increase their national testing capacities.
What makes the real-time RT-PCR technique so accurate for detecting SARS-CoV-2?
When you have to move from one place to another, obtaining quick results is essential. But speed should not compromise accuracy- as it’s crucial to identify people with the disease accurately. With accurate results, a COVID19 patient can be isolated to reduce the spread of the virus.
We have to consider both the analytical specificity and sensitivity of the real time PCR test to know its accuracy. The RT-PCR method assays itself to spot the viral RNA when it exists in the sample.
The analytical sensitivity of RT-PCR assays is still debatable. On the other hand, the clinical specificity and sensitivity determine the rate of :
- False positives- which refers to individuals who test positive but aren’t infected, and
- False negatives, meaning individuals who test negative but are infected with the virus.
Application of PCR
RT-PCR contains a high analytical and clinical specificity for SARS-CoV-2. It means this test produces fewer false-positive results. If a patient tests positive, we can conclude they have the virus.
The thing is, false positives are the result of technical errors or reagent contamination. One can avoid this by getting tested in a good laboratory that uses proper testing controls.
Keep in mind, the clinical sensitivity of RT-PCR for SARS-CoV-2 is around 70-80% . It means if you get a single negative test result, this doesn’t mean you don’t have the virus.
Possibility of false results in COVID-19 testing
Although RT PCR machines use primers that target the viral RNA that doesn’t change much, the mutations in the viral RNA sequence may lead to false or negative results by preventing proper primer binding.
Not every test kit is equal. Some have more sensitivity and specificity than others.
However, any RT-PCR assay shows results based on the sample collected.
Therefore, sample collection is the most significant factor in delivering false-negative results.
This can be many reasons, such as improper sampling technique, a mistake in storage or transport, and the type of specimen used. The timing of sample collection plays a vital role in the overall sensitivity of RT-PCR tests.
Explaining how real time PCR detects the COVID-19 virus
First, a healthcare professional collects a sample from the parts of the body containing the COVID-19 virus. The virus happens to be around a person’s nose or throat. The sample goes through multiple chemical solutions that remove proteins and fats and remains only with the RNA. This extracted RNA is a mixture of the infected person’s own genetic material and the virus’s RNA.
Then, the RNA is reverse transcribed to DNA, applying a particular enzyme. Healthcare professionals add extra tiny fragments of DNA complementary to certain parts of the transcribed viral DNA. If the virus exists in a sample, the fragments attract themselves to target sections. A few added genetic components help build DNA strands during amplification. In contrast, the others make the DNA and add labels to the strands to detect the virus.
In the next step, the mixture goes to the RT PCR machine. The machine produces temperatures that give heat and cool to the mix for triggering particular chemical reactions. It creates new or similar copies of the target gene of viral DNA. The cycle keeps on going on and on copying the target sections of viral DNA.
With each cycle, 2 copies become 4, and 4 copies become 8, and it goes on. A standard real time PCR technique typically goes through 35 cycles, meaning around 35 billion copies form from each strand of the virus present in the sample.
As new copies of the viral DNA take multiple forms, the marker labels attached to the DNA strands release a fluorescent reporter or dye. The machine’s computer measures this, and it appears in real-time on the screen. The computer detects the fluorescence amount in the sample every time.
If a particular level of fluorescence overshoots, it confirms the person has the virus. Scientists also keep an eye on how many cycles it takes to reach this level so that they estimate the severity of the infection.
So, in simple words, if a person has fewer cycles, it means the viral infection is mild; if more, then the viral infection is severe.
What do COVID-19 real time RT PCR test results mean?
If one gets a positive test result, it means they have SARS-CoV-2. Whether one has an asymptomatic infection or apparent symptoms- the infection is called COVID-19.
Many people get mild illnesses, and they recover quickly at home without medical care. However, one has to contact the healthcare provider if the symptoms get worse.
A negative test result shows that a person doesn’t have an infection with SARS-CoV-2. It is as per your collected specimen. However, one can have COVID-19 even if the test doesn’t detect the virus.
For instance, let’s say a person has recently become infected but doesn’t have symptoms yet. So, a negative test doesn’t mean the person is safe. He or she will get COVID-19 after the test and can spread the SARS-Cov-2 virus to others.
Better contact your healthcare provider, stay home and isolate yourself from others if you are COVID-19 positive. However, if the COVID 19 RT PCR test is negative, it’s still best to take steps to safeguard yourself and your family members from getting COVID-19.
Real-time RT-PCR test results time
One must obtain quick and accurate test results if one has to move from one place to another. In this case, real-time RT PCR is the best option as it takes approximately two to four hours, including the time for extracting and reverse transcribing the RNA. The specific real time PCR instruments are key to determine how quickly a particular lab can deliver results.
As you can see, real time PCR is highly effective. We hope we have covered many questions regarding this test. Feel free to share this informative blog with your family and friends.
You can book a real-time RT PCR test with us if you are in Seychelles.
Frequesntly Asked Questions
Scientists have to convert the RNA to DNA in order to detect a virus like the Covid-19 virus early in the body using real-time RT-PCR.
They have named this process reverse transcription. The reason for doing this test is because only DNA can be copied or amplified, making it a crucial part of the real-time RT–PCR process for spotting viruses.
Scientists have to amplify a particular part of the transcribed viral DNA multiple times. Amplification is essential. It supplies scientists with a large enough quantity of the target gene of viral DNA to verify that the virus is present in the body.
The real-time PCR technique is highly sensitive and can produce a reliable diagnosis in 3 hours. Keep in mind, laboratories may take between 6 to 8 hours. Real-time RT PCR is comparatively faster and contains a lower potential for errors or contaminations. The entire process happens within a closed tube, and it continues to be the most accurate method available.
The problem is that real-time RT PCR can’t detect past infections to understand the spread of the virus or its development. The reason is, viruses stay in the body for a specific window of time. Therefore, other methods are necessary to spot, track and study past infections.
Polymerase chain reaction real time or RT PCR is a variation of PCR. These two techniques implement the same method except that RT PCR comes with a reverse RNA transcription to DNA or RT for amplification.
To explain it in a simple language, a PCR test works on pathogens like bacteria and viruses, containing DNA for amplification. RT PCR is used for RNA, containing DNA for amplification. Both of these techniques can be performed in real-time, meaning results come immediately. If you go for other tests, you get results only at the end.
There is a considerable difference between these two types for COVID-19.
Polymerase chain reaction test detects the presence of the virus’s genetic material or its fragments while it breaks down. PCR data is quite reliable and accurate for detecting current infection in one’s body. Typically, it takes hours to perform PCR tests, but some are faster.
Antigen test detects bits of proteins that are on the surface of the virus called antigens. This test takes fifteen to thirty minutes. However, rapid antigen tests show accurate results if taken within a few days of your symptoms during the early stage, when the body has the largest amount of virus.
Real time PCR and the PCR test are very sensitive. These tests can detect fragments of the SARS-CoV-2 virus even if you have recovered from COVID-19. In this case, you may continue to test positive even if you can’t infect others with the SARS-CoV-2 virus. Not to mention, healthy people can become reinfected as well.
It would be best to discuss with your healthcare provider if you think you might have recovered from COVID-19 but still have symptoms.