RT-PCR - Reverse Transcription Polymerase Chain Reaction

The covid-19 pandemic has affected the lives of people and brought many changes. These include the economic crisis to the shortage of healthcare services. Hence the solutions to control the rise of covid-19 cases are a must. Testing techniques and preventive measures play a major role in reducing covid-19 cases in the country. One such testing method is RT-PCR Reverse Transcription Polymerase Chain Reaction. It is one of the most accurate techniques for the detection of the virus.

It is a real-time test; therefore, it does not consume much time for the detection of the virus. Additionally, it only detects the covid-19 virus. Hence cannot be used for other diseases.

What is RT-PCR?

RT-PCR is a real-time technique used to detect a specific genetic material. It is a nuclear-derived method that is useful to detect disease in pathogens and viruses. RT-PCR is a highly sensitive technique that is specific to the covid-19 disease. Conventionally, a different technique was used to conduct the test, which used radioactive isotope markers. Nowadays, a more evolved technique is used, which uses special markers like fluorescent dyes. The new method provides immediate results while the process is still in progress. However, the conventional test takes more time, and the result can be obtained only when the process terminates.

Today, real-time RT-PCR is widely used methods of testing or detecting the covid-19 infection. It has helped in the testing of diseases such as the Ebola virus and Zika virus. Many countries use real-time RT-PCR for the detection of other diseases. However, some countries still need to adapt this method for faster and efficient testing, eventually, reducing the risk of the infection.

What is a virus?

A virus is a package of genetic material which can be either DNA (Deoxyribonucleic Acid) or RNA (Ribonucleic Acid). This package of genetic material is surrounded by an envelope.

What is Genetic Material?

Genetic material is a substance that is inherited by offspring and stores all the necessary information for the growth and development of that organism. It generally contains information that is specific to a particular organism. This information is required to direct the protein synthesis of the organism. Any organism is built based on this data, and it also acts as the blueprint for copying. An example of the genetic material is deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). These are explained below -

DNA (Deoxyribonucleic Acid) - DNA is a molecule that holds the information necessary for reproducing the offspring of any animal. It is a two-stranded molecule that stores the characteristics of any organism. It is also known to store the genetic code or blueprint of an organism.

RNA (Ribonucleic Acid) - RNA transmits the information to proteins that are necessary for the synthesis and development of an organism. These are essential for the organism to be alive and for its overall growth. RNA is a single-stranded molecule; copying and transcribing are also some of its functions.

Viruses such as SARS-CoV-2, which is the cause of covid-19 disease, have only RNA structure as their genetic material. Thus, they use RNA to program the cell in such a way that they start producing more viruses. Eventually, the cell becomes a factory for virus production and takes control of the cell.

In real-time RT-PCR, it is required to convert the RNA into DNA to detect the coronavirus in the body. This process is known as reverse transcription. It is essential because RNA cannot be copied, unlike DNA. Hence, in real-time RT-PCR, RNA is transcribed into DNA, and this is the most important step of detecting viruses.

Scientists also amplify the DNA to have a large amount to detect the presence of viruses. However, only the specific or targeted sections of the transcribed DNA are used for amplification. DNA amplification provides the size required for effective detection of the viruses present. It results in a more accurate result of the test.

Working of real-time RT-PCR and Detection of Viruses

1. Collection of samples - Sample from nose and throat is collected for testing as this is the most prone area for the virus collection.
2. Chemical treatment - The sample is treated with chemicals to remove fats and proteins and extract RNA. Usually, this RNA will contain only the genetic material of the person. However, if the person is positive for covid-19, the RNA will contain the genetic material of the virus along with that of the person.
3. Transcription of RNA and testing - The RNA is transcribed to DNA using a special enzyme. While the transcription is in progress, scientists insert the fragments of DNA into the transcribed DNA. If the RNA extracted from the sample contains the virus, then the fragments attach themselves to the target sections of the virus. Some fragments are used for creating DNA during amplification. However, other strands are used as marker labels to detect the virus.
4. Placing mixture in the RT-PCR machine - The mixture is placed into the RT-PCR machine. It completes its target by moving the mixture through various temperatures ranging from hot to cold. The purpose of the machines is to start a chemical reaction in the mixture through the cycling of the chemicals. After the reaction, new and identical copies of the viral DNA are formed.
5. Repetition of the cycles - The cycle in the machine continues repeating itself. It leads to the copies of the DNA, which multiply from two to four, four to eight, and so on. Generally, a real-time RT-PCR setup undergoes around 35 cycles of the machine. In this process, 35 billion copies are generated from each strand of DNA.
6. Final Detection - After the creation of multiple new copies, the marker labels are intended to attach to the DNA. This process is followed by the release of fluorescent dye. The computer system is used to detect the amount of fluorescent dye in the sample. The readings after each cycle are recorded through continuous monitoring of the machine. If the level of fluorescent passes a certain set mark, the presence of the covid-19 virus is confirmed. After scientists get the confirmation, they evaluate the severity of the disease through the number of cycles of the machine. The fewer the number of cycles in reaching the set mark of the fluorescent dye, the more severe will the impact of disease on that person.

Steps to follow after an RT-PCR Test

For the person who tests positive in RT-PCR -

The healthcare provider will guide the person and prescribe appropriate medicines and the necessary measures that must be followed after testing positive. These are generally provided to limit the spread of infections to other healthy people.

For the person who tests negative in RT-PCR -

A negative result of an RT-PCR test assures that the person did not have a covid-19 infection at the time of testing. However, if someone has symptoms 3-4 days after the RT-PCR test,they must take the RT-PCR test again. After which, the person may consult a doctor according to the results of the test.

Reasons to use RT-PCR

1. Sensitivity - RT-PCR is a highly sensitive technique that is specific to the covid-19 disease.
2. Reliable Diagnoses - RT-PCR provides a very reliable diagnosis of the infection, and the chance of error in the report is less as compared to the other testing method.
3. Consumes less time - RT-PCR is less time-consuming as it requires three to four hours to conduct and find the test results. However, labs usually take around six hours to detect the infection.
4. Accuracy - RT-PCR is one of the most accurate techniques to find the infection in the body. The reason for its accuracy is that it is conducted in a closed tube. Hence the error rate is much lower compared to the other method of detecting infectious disease.

Other Covid-19 Tests

1. Rapid Antigen Test -
   • Any foreign matter that triggers the immune system is known as the covid-19 antigen. This test detects the antigens or viral proteins present in the body. The rapid antigen test is faster as compared to any molecular test. However, the major drawback of this test is that the probability of error is more in the negative result of this test.
   • Sample of nasal or throat swab is taken and tested using the rapid antigen equipment.
   • The time required for the results is less as compared to all other covid-19 tests. The results take approximately 1 hour. Hence, it is known as a rapid antigen test.
   • Another test is required to be done to confirm the results if tested negative. However, it has high accuracy for positive results.
   • It is useful to diagnose coronavirus in the sample.
   • There are chances of missing out on an active coronavirus case in a rapid antigen test.
   • Rapid antigen test costs about Rs. 400 to Rs. 500.
2. Covid-19 Antibody Test
   • This test finds out whether your immune system is exposed to the virus or not. It is also known as a serology test, and it detects covid-19 antibodies in blood using screening of the patient. The antibodies determine that the person is previously infected with the virus causing covid-19 infection. It determines the covid infection without testing the active covid-19 virus in the patient's body.
   • The samples taken are blood samples which are later sent for testing.
   • The results of the antibody test usually take 1-3 days. However, sometimes it is delivered on the same day of testing.
   • Though the accuracy of this test is good enough, sometimes another test or repetition of the antibody test is required to confirm the results.
   • It is an effective technique to find out covid-19 infection in the past or now through the antibodies in the body.
   • It does not show active coronavirus in the body, which is its biggest disadvantage.
   • It is not a very expensive test and costs around Rs. 500 to Rs. 600.

The PCR Technique

The full form of PCR is Polymerase Chain Reaction. It is one of the most popular techniques used to detect the spread of diseases caused by pathogens. It has played a vital role in detecting and controlling diseases like the Ebola virus and swine flu.

Similarities and Differences between PCR and RT-PCR

Similarities -

Both PCR and RT-PCR use the same technique to identify the disease except the last step of RT-PCR. Both the methods are real-time and reliable to detect the infection caused by diseases. The results of both tests can be obtained immediately.

Differences -

RT-PCR has an additional step in the detection technique. This step is reverse transcription of RNA used for amplification. Hence the major difference is that the PCR is used for detecting infections caused by pathogens that already have DNA. However, RT-PCR is the technique that is used for pathogens containing RNA. Hence, it is required to transcribe the RNA into DNA in RT-PCR for amplification.

Advantages and Disadvantages of RT-PCR

Advantages

1. RT-PCR is believed to be a well-documented and effective technique. It is approved by various scientists and medical staff.
2. RT-PCR is effective as extensive research is conducted. Hence it can be used to test Covid-19.
3. It detects the infection of the disease and can determine if the person is positive or negative for the infection.

Disadvantages

1. RT-PCR can miss the patients who have had the infection and have recovered as it detects only if the virus is present.
2. Through RT-PCR, the virus may be detected in the sputum or nasopharyngeal swab but not in both at the same time. It detects the virus infection, which may vary from person to person.
3. RT-PCR can only detect the symptoms and infection through covid-19. It doesn't show any signs of other diseases.
4. There is a slight possibility of error in the test. The ideal method of testing to obtain the appropriate result is to prefer a CT scan with RT-PCR. Both clinical features and RT-PCR are most accurate in determining the result of a person.

Results of RT-PCR Test and Related Issues

The results of the RT-PCR test are accurate when compared to the other methods of detecting the infection. It is a real-time test; therefore, it does not consume much time for the detection of a virus. However, there is still a chance of wrong results for certain cases. There are many reasons for the wrong positive or wrong negative results of the RT-PCR test. The most common reason for the negative report of a positive patient is that only upper respiratory tract samples are tested. In order to avoid this error, the lower respiratory tract sample must also be tested at different points of time. The ideal method of testing to obtain the appropriate result is to prefer a CT image with RT-PCR. Both clinical features and RT-PCR are most accurate in determining the result of a person.