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All you need to know about the RT-PCR test
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All you need to know about the RT-PCR test

Widely deployed during the COVID-19 pandemic, the reverse transcription-polymerase chain reaction test can detect the presence of pathogens in a sample within a short time
The RT-PCR test widely deployed during the COVID-19 pandemic can detect the presence of pathogen in a short time
Photo by Anantha Subramanyam K/Happiest Health

If you’ve been tested during the COVID-19 pandemic, then you’re no stranger to the real-time RT-PCR (reverse transcription polymerase chain reaction) test — a molecular assay that detects the presence of certain viruses (including the coronavirus) from a swab sample within hours.

PCR (polymerase chain reaction), a type of nucleic acid amplification test through which one can make millions of copies of a specific DNA sequence, is the foundation of this revolutionary technology.

Dr Sushma Krishna, consultant microbiologist at Sagar Hospitals, Bengaluru, says that RT-PCR, an accurate and reliable test, is considered the ‘diagnostic tool of the millennium’. According to the Infectious Diseases Society of America (IDSA), when the COVID-19 pandemic began, RT-PCR tests were “the first to be developed and widely deployed.”

What makes the RT-PCR test reliable?

Dr Ambica R, head of the department of microbiology at Bangalore Medical College and Research Institute (BMCRI), explains how the genetic material of viruses invades human cells. “When viruses like SARS-CoV-2 enter the human body, they invade the cells of their target organ or organ system and multiply. Subsequently, they cause local or systemic manifestations of an infection.”

Putting it in simple terms, Dr Laxman Jessani, consultant infectious diseases specialist at Apollo Hospitals, Navi Mumbai, says that PCR involves the conversion and amplification of a small piece of genetic material of the suspected virus to detectable levels. However, Dr Krishna informs that the RT-PCR test involves a few additional steps.

What happens in the lab?

During PCR, the viral genetic material extracted from the samples of suspected individuals is mixed with enzymes and other solutions. This mixture is then fed into a machine called the thermal cycler, more commonly known as the PCR machine, explains Dr Krishna.

“This machine will then amplify the genetic material in a small test tube,” she says, adding that some viruses have RNA (ribonucleic acid) as their genetic material (like SARS-CoV and hepatitis C virus) and are referred to as RNA viruses. On the other hand, DNA viruses — having DNA (deoxyribonucleic acid) as the genetic material — include smallpox viruses, adenoviruses, etc.

Dr Krishna lists the three key steps involved in a PCR test:

  • Sample collection: In the first step, sufficient serum or plasma sample of the person is collected by a healthcare provider. In cases of respiratory infections like H1N1, the flu and COVID-19, the sample is collected from the nose or throat using a sterile swab. It is then sealed in a transport media tube and sent to a laboratory.
  • Sample processing: At the lab, the genetic material of the virus is extracted from the sample. During this step, reagents and enzymes capable of synthesizing a new DNA strand are used, and a master mix is prepared. Each heating and cooling cycle in the PCR machine amplifies the amount of the targeted genetic material in a 96-well plate (plastic, heat-resistant multi-well microplates designed for use inside PCR machines or real-time thermocyclers). One of the reagents in the tube produces a fluorescent light, indicating the presence of the virus in the sample. Once amplified enough, the PCR machine detects this signal. Special software is then used to interpret the signal as a positive test result.
  • RT-PCR: In this case, an additional step in the beginning involves the conversion of RNA into complementary DNA (cDNA) through an enzyme called reverse transcriptase. This applies to RNA viruses like SARS-CoV and HCV.

Dr Krishna says that there are other types of PCR (such as multiplex PCR) where the presence of multiple pathogens can be detected simultaneously in a single step.

Interpreting RT-PCR test result and Ct value

Dr Jessani says, “A positive [RT-PCR] test result means that the process has detected certain genes of the particular virus in the individual suspected of infection.”

The Ct (cycle threshold) value in an RT-PCR test report helps roughly determine the viral load and estimate the transmission potential of the virus, says Dr Krishna. “If the Ct value is low, it means fewer cycles were required in the PCR machine to detect the virus, implying a higher viral load and transmissibility. A higher Ct value, on the other hand, suggests a lower viral load and transmissibility,” she adds.

Takeaways

  • The RT-PCR test is a reliable diagnostic tool that can detect the presence of a pathogen (like a virus) from a swab sample within hours.
  • Widely used during the COVID-19 pandemic, the process involves converting and amplifying a small piece of genetic material of the suspected virus to detectable levels.

Share Your Experience/Comments

2 Responses

  1. In many cases, the COVID rapid cards are found negative for COVID but RT PCR is positive. Why?

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