As the second wave of the COVID-19 pandemic encompasses India, with more than 3 lakh people developing coronavirus infection in a day, the need of the hour is to ramp up testing among the general public. Spotting those who are afflicted with COVID-19 promptly, ensures that those individuals can be isolated, quarantined at once and given proper medical treatment depending upon the severity of their symptoms, in addition to halting community spread of the disease. When it comes to healthcare protocols to identify COVID-19 positive cases, the RT-PCR test is considered to be the gold standard of diagnostic assays with regards to coronavirus infection.

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What Is The RT-PCR Test?

RT-PCR stands for a reverse transcriptase-polymerase chain reaction. It is a common validated diagnostic test to recognise if viruses transmitted via the respiratory pathway such as SARS-CoV-2 are present in a person. This is done by directly noticing certain specific viral components characteristic of SARS-CoV-2 in the respiratory samples obtained from the individuals, such as a nasal swab and throat swab.

What Is The Principle Behind The RT-PCR Test?

The RT-PCR technique is a laboratory protocol used to identify the presence of RNA and the quantity of this genetic material, in a sample. This pioneering scientific procedure was developed by Kary Mullis, a renowned American biochemist, in the year 1983 and he was awarded the Nobel Prize in 1993 for his groundbreaking invention. He shared this Nobel Prize with another distinguished British-Canadian biochemist, Michael Smith, who designed the protocol of site-directed mutagenesis, a very useful technique to modify DNA sequences to aid in investigating the structure and biological functions of DNA, RNA, proteins.

The core genetic matter in viruses, including the SARS-CoV-2 strain of coronavirus, is RNA i.e. Ribonucleic Acid. To accurately spot this viral RNA in a sample of a person’s respiratory secretions, lab technicians and medical researchers require the complementary DNA (Deoxyribonucleic Acid) segment to this RNA strand and in a large quantity. This is what the protocol of RT-PCR achieves – the conversion by an enzyme i.e. reverse transcription of viral RNA to complementary DNA or cDNA sequences, which are then amplified by the PCR i.e. Polymerase Chain Reaction, to precisely detect the presence of viral RNA.
Woman undergoing RT-PCR test

COVID-19 RT-PCR Test Procedure:

The RT-PCR Test for COVID-19 is usually performed in a hospital, doctor’s clinic, diagnostic centre and even in community health camps, terminals of airports, or at home, to identify if SARS-CoV-2 viral strain is present in the individual.

COVId-19 RT-PCR Test is a simple, straightforward procedure wherein the doctor, nurse, lab pathologist or certified healthcare professional inserts a clean swab into the nose and slowly scrubs it across the nasopharynx, the portion in the roof of the mouth. Likewise, another swab is inserted into the throat and both samples are placed in separate test tubes containing a solution. This filters out excess proteins, fats in the patient’s nasal swab and throat swab collections and the resultant sample contains only the person’s DNA genetic material and if infected with COVID-19, the SARS-CoV-2 coronavirus RNA matter as well.

These nasopharyngeal cultures i.e. distilled nasal swab and throat swab samples are made to undergo reverse transcription and then placed in the RT-PCR machine. This processes the samples in repetitive cycles of hot and cold temperatures, normally up to 35 – 40 rounds, which generate more copies of the cDNA. These are represented through binding fluorescence markers and showcased real-time on a computer screen, to finally identify if SARS-CoV-2 viral RNA is present in the sample. The results of the COVID-19 RT-PCR Test are usually provided 2 – 3 days after collecting the nasal and throat swab samples.

Results Of The RT-PCR Test:

The RT-PCR Test results inform the individual if they COVID positive or COVID negative, by confirming the presence of SARS-CoV-2 viral RNA and the CT value calculated in the patient’s nasopharyngeal sample.

What Is Meant By CT Value?

CT value or cycle threshold value refers to the number of cycles required in the polymerase chain reaction, to amplify the complementary DNA obtained from reverse transcription of viral RNA, to a sufficiently noticeable level, to accurately spot viral segments of SARS-CoV-2, if present.

Hence, the lower the CT value, the person has a high viral load, since it means that just a few PCR cycles were adequate to amplify the SARS-CoV-2 viral RNA. Similarly, a higher CT value indicates a lower viral load, as several cycles of PCR were needed to amplify the SARS-CoV-2 viral RNA present in the person’s swab sample.

How Accurate Is The RT-PCR Test In Detecting COVID-19 Infection?

The RT-PCR Test for COVID-19 is by far the most accurate testing procedure utilised all over the world to identify if a person is positive for coronavirus infection, widely regarded as the gold standard of COVID-19 diagnostic tests.

However, it sometimes indicates a person to be positive even if they have completely recovered from COVID-19 due to remnants of SARS-CoV-2 viral strain in the individual’s swab samples. Moreover, it is not known if the RT-PCR Test can detect the mutated SARS-CoV-2 strains with high sensitivity and accuracy and more scientific research is needed in this regard.