How Real Time PCR Technique is used to detect covid

Real-time PCR is a diagnostic technique that is widely used to detect the COVID virus. Before we get into how this technique works or how useful it is, let’s learn a thing or two about the virus and genetic material that the machine detects.

If you have symptoms of COVID-19 or want to fly out of Seychelles- you must get a test done. RT PCR tests are available at Seychelles Medical. All you have to do is book online– and we will get in touch with you as soon as possible.

Now, let us understand how RT PCR tests work and how they detect COVID-19.

What is a virus? What is genetic material?

A virus comprises the microscopic package of genetic material enclosed by a molecular envelope. This genetic material can be either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA).

DNA is the two-strand molecule in every organism, such as plants, animals, and viruses, and any living organism that holds its genetic code or blueprint.

RNA is a one-strand molecule that copies transcribes, and transmits genetic code sequences to proteins. This is so that the body can synthesize and do the functions that keep organisms alive and developing. Different variations of RNA perform the copying, transcribing, and transmitting.

Some viruses like the coronavirus (SARS-CoV-2), that causes COVID-19, contain RNA only.

That means that they depend on infiltrating healthy cells to multiply and live. Once they get inside the cell, the viruses use their own genetic code, RNA, when it’s about the COVID-19 virus.

Scientists convert RNA to DNA in the RT–PCR process to detect the COVID-19 virus in the body.

This process is called ‘reverse transcription. Scientists do this because just DNA can be copied or amplified. This is a key part of the real time PCR instrument for detecting viruses.

Scientists amplify a definite part of the transcribed viral DNA thousands of times. Amplification is essential so that rather than trying to spot a minute amount of the virus among millions of target gene strands, scientists have a big enough quantity of the viral DNA’s target sections to precisely confirm if the virus is present.

How does the real time PCR machine work with the COVID-19 virus?

The medical worker collects a sample from the body part where the COVID-19 virus is present, such as the patient’s throat or nose. Lab workers then treat the sample with some chemical solutions to remove substances such as fats and proteins and extract only the RNA present. This sample RNA is a mixture of the person’s gene expression and, if present, the virus’s RNA.

The RNA is reverse transcribed to DNA with the help of a specific enzyme. After that, scientists add extra short DNA fragments to complement particular parts of the transcribed viral DNA.

If the virus is there in a sample, the fragments attach themselves to target segments of the viral DNA. Some additional genetic fragments help build DNA strands during amplification. On the other hand, other fragments facilitate building the DNA and adding marker labels to the strands.

This is how scientists can detect the virus in the lab.

A look inside the machine- what is RT PCR test 

The mix is then placed in an RT–PCR data machine. This machine cycles through temperatures that heat and cool the mix to trigger specific chemical reactions to create new and identical copies of the target sections of viral DNA. The cycle keeps repeating and continues copying the viral DNA’s target sections.

Every cycle doubles the previous number: two copies turn into four, four become eight, and so on. A typical real-time RT–PCR set-up goes through 35 cycles. By the end of the process, a single virus strand in the sample can generate 35 billion new copies of the viral DNA sections.

As new duplicates of the viral DNA sections build, the marker labels attach to the DNA strands. Then they release a fluorescent dye, measured by the machine’s computer and represented in real-time on the screen. The PC has a fluorescent reporter that tracks the amount of fluorescence in the sample after every cycle.

Once a certain level of fluorescence surpasses, you can tell that the virus is present. Scientists also track how many cycles it takes to get on this level to estimate the severity of the infection: the lesser the cycles, the more intense the viral infection is.

Why use a real time PCR machine? 

The real-time RT–PCR technique is highly specific, sensitive, and can deliver a consistent diagnosis in less than three hours. The laboratories take on average six to eight hours. In contrast with different available virus isolation methods, the real-time RT–PCR test is significantly faster and has a lower chance of contamination or errors.

Scientists can carry out the whole process in a closed tube. It is the most accurate process available for detecting the COVID-19 virus.

However, real-time RT–PCR technology cannot detect past infections. This data is important for understanding the growth and spread of the virus, as those are only present in the human body for a specific period. Having data over past infections is important because it helps us identify

What is PCR? How is it different from real-time RT–PCR? 

Real time PCR is a variation of transcription polymerase chain reaction, commonly known as PCR. The two techniques go through the same process except that RT–PCR has one more step of reverse transcription polymerase chain of RNA to DNA to allow for amplification. This simply means that polymerase chain reaction real time is mainly for viruses and bacteria that already have DNA for amplification.

Scientists apply RT–PCR to RNA that must be transcribed to DNA for amplification. Both of these are real-time techniques. This means results are visible immediately, while when used ‘conventionally,’ results are visible only at the end of the reaction.

PCR is among the most popular diagnostic tests to detect pathogens and viruses that cause diseases such as African swine fever, Ebola, and foot-and-mouth disease. Since the COVID-19 virus contains just RNA, it is best to detect it in real-time or in conventional RT–PCR.

Pros and Cons of Using qPCR in Quantification and Detection and Pathogens 

Since PCR can amplify specific DNA fragments, it has wide applications in pathogen diagnostics. With the increasing amount of sequencing data available, it is possible to design qPCR assays for each microorganism (groups of microorganisms, etc.). The main benefits of qPCR are that it delivers fast and high throughput quantification and detection of target DNA sequences in multiple matrices.

The lower time of amplification is due to the simultaneous amplification and picturization of newly formed DNA amplicons.

Moreover, qPCR is safe when it’s about avoiding cross contaminations. This is because no further manipulation is necessary for samples after the amplification. Other benefits of qPCR include an extensive dynamic range for quantification (7–8 Log10) and the multiplexing of numerous targets’ amplification into a single reaction.

Multiplexing is essential for quantification and detection in diagnostic qPCR assays that depend on internal amplification.

Real time PCR meaning- how does it work 

Real time PCR occurs in a thermal cycler with the capacity to illuminate every sample with a beam of light of at least one particular wavelength and detect the fluorescence emission by the excited fluorophore. The thermal cycler can also rapidly heat and chill samples. It takes advantage of the physicochemical properties of DNA polymerase and nucleic acids to give you the results.

The PCR process consists of a series of temperature variations that are repeated 25–50 times. These cycles usually comprise three stages:

• At 95 °C, the separation of the nucleic acid’s double chain.
• The second stage is at a temperature of around 50–60 °C, which does the primers’ binding with the DNA template.
• The third stage, between 68–72 °C, facilitates the polymerization drawn out by the DNA polymerase.

Because of the small size of the fragments, the last step is omitted in this type of PCR. This happens as the enzyme can replicate the DNA amplicon during the change between the denaturing stage and the alignment stage.

Four-step PCR technique for real time PCR 

Additionally, the four-step PCR measures the fluorescence during short temperature phases. Those last only a few seconds in each cycle, having a temperature of, for instance, 80 °C, to neutralize the signal triggered by the presence of primer-dimers every time a non-specific dye is used.

The temperatures and timings for each cycle depend on various parameters, like the enzyme that synthesizes the DNA, the concentration of divalent ions, and (dNTPs) deoxyribonucleotide triphosphates in the reaction and the primer’s bonding temperature.

What are the PCR steps? 

All the real time PCR experiments happen through the single PCR process mentioned below-

Step 1: Denaturation 

Just like in DNA replication, this involves separating the two strands in the DNA double helix.

The separation occurs by raising the temperature of the mix. Denaturation breaks the hydrogen bonds between the complementary DNA strands.

Step 2: Annealing 

Primers bind to the target DNA segment and initiate polymerization. This only occurs once the temperature of the solution lowers. One primer binds to each strand.

Step 3: Extension 

New strands of DNA builds using the original strands as templates. The DNA polymerase enzyme joins DNA nucleotides together. This enzyme is usually Taq polymerase, an enzyme isolated from a thermophilic bacteria known as Thermus aquaticus. The sequence of nucleotides in the original DNA determines the order in which the free nucleotides add on.

By the end of one cycle of PCR, two double-stranded segments of target DNA are formed as PCR products, each containing a newly made strand and an original strand.

The cycle repeats multiple times (usually 20–30) as most processes of PCR need large quantities of DNA. It takes just 2–3 hours to get a billion or so copies.

Conclusion 

PCR is a widely used test today. It is the gold standard test for detecting COVID19. Seychelles Medical can carry out RT PCR tests- along with other tests that can help you get an accurate diagnosis. Book your test today!