What is Rt-PCR Test for Covid

Coronavirus or SARS-CoV 2 causes the Covid-19 disease, which is now a worldwide pandemic. One of the primary modes for combating this pandemic is rapid identification of the infected persons so that subsequent treatment can begin fast, and they can be quarantined before spreading it to others.

Symptoms that should lead to testing:

  • Fever
  • Chills
  • Loss of breath
  • Respiratory problems
  • Fatigue
  • Weakness
  • Muscle pain
  • Loss of smell and taste sensations
  • Headache
  • Body ache
  • Sore throat
  • Lung congestion
  • Running nose
  • Diarrhoea
  • Nausea

Apart from these, some infected persons may not show any signs of these symptoms, but can spread the disease nevertheless, so it is crucial to undergo testing even for these asymptomatic persons.

Some IDSA (Infectious Disease Society of America) guidelines for undergoing testing are as follows:

  • If anyone has been in close contact as within 6 feet of an infected person and exposed for more than 15 minutes.
  • If one has participated in any public event like mass gatherings, marriage, or any party, travelled recently, or has been confined within an enclosed area with large crowds.
  • Before any major surgery or during immunosuppressive therapy, as they are more at risk.
  • Before any organ transplantation or stem cell surgery.
  • Before and after childbirth.
  • Repeat testing in suspected individuals even when primary test results are negative.

Types of testing for Covid-19

There are multiple diagnostic tests and antibody test types for Covid-19.

Diagnostic tests are important for detecting active infection in a person. These are mainly 2 types:

  • Molecular tests, like the RT-PCR test for Covid-19.
  • Antigen-based rapid test for Covid.

The antigen-based test identifies typical surface antigens present within the sample using immune-chromatographic methods. Though it is effective for identifying acute cases and is handier as it can be performed at home, the sensitivity is lower than molecular methods, about 30–40 % less. Hence, it can’t be used as a confirmatory test for Covid diagnosis and needs to be followed by molecular ones.

Sample collection

Samples are typically collected by using a cotton swab, a long tube with sterile cotton attached at one end enclosed within a capped sterile tube.

The samples tested for the Covid-19 Rt-PCR test, primarily, are:

  • Anterior nares (Nasal) – Samples collected from the nostrils.
  • Mid-turbinate – Samples from deep within the nose.
  • Nasopharyngeal – Further deeper inside the nose, adjoining the throat.
  • Oropharyngeal – Deep inside the throat in the pharyngeal region, just below the mouth.

Saliva samples are also collected by the patient spitting into a sterile tube.

What is the Rt-PCR test for Covid-19?

Rt-PCR stands for Reverse Transcriptase-Polymerase Chain Reaction. It is a highly sensitive, quantitative test for detecting even trace amounts of any viral or bacterial genetic material within the sample.

This test is the gold standard for Covid-19 virus detection and has been approved for use since February 2020.

The basic steps of an Rt-PCR test are as follows.

Extraction

The sample collected is extracted to isolate the genetic materials, typically done by adding chemicals and peptides that dispose of any proteins or chemical particles from the sample leaving behind pure genetic materials only like RNA or DNA for analysis.

Reverse transcriptase

This is an enzyme that is capable of transcribing complementary DNA strands from a single mRNA strand. This cDNA strand is then amplified and detected using PCR (Polymerase Chain Reaction) technique. This test typically can detect even a minuscule amount of RNA (as SARS-CoV is an RNA-containing virus) present within the sample and is highly specific.

The basic ingredients used for PCR set-up are as follows:

  1. A DNA template strand to bind to the target region of DNA and then to amplify. For Covid-19 generally, the ORF1ab gene is used as a target. This is a SARS-COV gene found in its nucleocapsid region.
  2. A heat-stable DNA polymerase enzyme that can synthesise new DNA strands from the mother cDNA. Generally, heat-resistant Taq polymerase isolated from thermophilic bacteria residing in volcanic regions is used for this purpose.
  3. Two types of DNA primers that are complementary to both the strands of the target DNA, as DNA polymerase can only bind and amplify double-stranded DNA only. Primers are single-stranded DNA fragments that are smaller in size but site-specific. Primers specific to target DNA strands are prepared in biochemical laboratories and supplied to customers.
  4. Deoxyribonucleoside triphosphates or dNTPs, are the units of DNA. These dNTPs are polymerized by polymerase enzymes into the new DNA strands.
  5. A suitable buffer solution for providing an optimal chemical atmosphere for the reactions.
  6. Magnesium (Mg2+) or Manganese (Mn2+) cations

PCR reaction is carried within small Eppendorf tubes of approx 0.5 mL volume, within a thermal cycler. This cycler can control accurate temperatures within the reaction centre and heats and cools the tubes according to the required steps.

PCR reaction consists of repeated extreme changes in temperatures from 20 to 40 thermal cycles for a certain amount of time. These changes depend on several factors like the temperature sensitivity of the enzymes used, melting temperature (the temperature at which the double-stranded DNA separates from each other) of the DNA primers, and concentration of the Mg/Mn cations and the dNTPs.

The steps of PCR or polymerase chain reaction are as follows:

Initialization: This step includes heating the reaction chamber to a high temperature of 94–98 °C for 10 minutes to activate the Taq polymerase enzyme that reacts at this optimum temperature. This initiates the PCR.

Denaturation: This is the first step of the thermal cycle. The reaction chamber is heated to 94–98 °C and kept at it for 20 – 30 seconds. At this stage, the template DNA strands (obtained from the Reverse transcriptase stage) melt, separating from each other due to breakage of the Hydrogen bonds connecting the complementary bases resulting in two fragments of single-stranded DNAs.

Annealing: At this step, the temperature is dropped to 50–65 °C and kept at it for 20–40 seconds. As the DNA strands cool down, the primers bind to their complementary target site of the denatured DNA template strands. The temperature and holding time are very crucial, as they should facilitate the perfect hybridization of the primer with the template. The optimal temperature is generally 3–5 °C below the melting temperature of the primer DNA. A temperature lower than that will result in erroneous binding resulting in wrong DNA synthesis, whereas a higher temperature will prevent any binding at all.

Extension or elongation: This temperature depends upon the polymerase enzyme used for DNA synthesis. Taq polymerase acts at an optimum temperature of 75–80 °C (preferably 72° C). The time for complete elongation of the cDNA strand varies on the type of polymerase enzyme used, as well as the length of the target DNA. In general, it is supposed that a single polymerase enzyme can bond about a thousand bases every minute, and under the perfect conditions at each elongation step the total nos. of template DNA strands is doubled. Therefore, after the completion of each cycle, the starting amount of DNA is to be doubled exponentially.

Final elongation: After completion of the required thermal cycles, at this penultimate step, the temperature is kept at 70–74° C for 5 to 15 mins. This step is conducted to make sure any leftover single-stranded DNA can be extended completely.

Final hold: At the final step, the reaction centre is finally cooled to 4–15° C and can be stored temporarily.

Real-Time Rt-PCR

The Covid-19 Rt-PCR test has a real-time variation. Real-time Rt-PCR is a modified version for quantifying the amount of DNA amplified after each cycle of PCR using spectrofluorometry. In the reaction mixture, a site-specific probe is added; the most commonly used is the TaqMan probe. It has a fluorescent molecule and a quencher molecule added at two ends. The quencher is a molecule that absorbs any fluorescence from the fluorophore and hence the probe is undetected at the primary stage.

During subsequent steps, the probe attaches itself to the target DNA, but during the elongation step, the probe is denatured by the DNA polymerase, freeing the fluorophore from the quencher. Hence, we can see that as the cycles of PCR progress, more DNA is amplified and more fluorophore is accumulated. This fluorescence is detected and quantified using a spectrofluorometer and thus helps in quantitative analysis.

Time duration for Rt-PCR test result

Typically a Covid-19 test result is available within 24 hours of sampling, but the duration varies depending upon the sample load, the time taken by the sample to reach the testing site, and indication of false test results.

Interpretation of Covid-19 Rt-PCR test results

Studies show the Rt-PCR test gives the most accurate results when tested within 3 days after infection as the viral load slowly decreases after a week of the infection.

A positive test should result in the immediate isolation of the infected person and the administering of proper medication, as advised by a doctor. Most people, like those with asymptomatic infections or those with moderate symptoms, can heal and recover at home without medical intervention.

But one must seek immediate medical care in case of the following symptoms:

  • Breathing difficulty
  • Confusion
  • Chest pain
  • Blue coloration of lips or face
  • Heavy chest pressure
  • Feeling sleepy and drowsy

A negative test result indicates that the person wasn’t infected before the sample collection, but it in no way confirms subsequent infection and hence one should follow strict protocols like social distancing and wearing masks all the time.

What is meant by Ct values for RT-PCR test results?

The Ct-value in an Rt-PCR test result stands for ‘cycle threshold’ of the coronavirus and is an indication of the viral load within the patient. With the emergence of various mutated variants of the SARS-CoV 2 virus and its proximity to human coronavirus, Ct-value is crucial for disease diagnosis.

A Ct-value less than 35 is considered to be a positive result whereas those higher than 35 are negative. Ct-value indicates the numbers of cycles of RT-PCR needed to be run to detect a positive coronavirus. Hence, a cut-off value of 35 indicates a total of 35 cycles of RT-PCR is run before any detectable amount of viral genetic material is found.

A lesser value indicates a higher concentration of viral genes present within the sample and hence deems the person as highly contagious.

Importance of Ct-value

The Ct-value can be indicative of the transmission potential of the person tested. In simple words, a higher concentration of viral materials in one’s throat and nasal cavity means he will more easily spread the virus than those with a lesser value. Hence, it is important in assessing the prevalence of infection in a particular region.

Also, studies indicate that there is no direct link between a lower Ct value with the mortality rate or the severity of the coronavirus infection. It is linked only with the time of infection and onset of the symptoms and infectivity, so there is no need to panic when one gets a lower Ct-value in a Covid-19 test result.

Can the Covid-19 Rt-PCR test differentiate between different mutated variants of SARS-CoV2?

The SARS-CoV2 is a highly mutable strain of the virus and has resulted in various genetic mutated strains, like the Omicron and delta variants.
Presently, the RT-PCR for Covid-19 can only identify the SARS-CoV2 virus broadly and no authorised tests kits to identify mutated strains are available yet.

The mutated species show different serological, antigenic, and genetic properties and can impact the test results. So, it is advised to consider the patient’s history, clinical symptoms, and epidemiological impact even if the test results are negative.

Repeated testing with alternative genetic targets of Covid-19 as authorised by the FDA, is to be done as different variants may contain different genetic markers and can remain undetected.

Conclusion

Rapid and accurate identification of Covid-19 is the primary tool for decreasing the coronavirus pandemic.

One should always be alert by avoiding crowded gatherings and using face masks and monitoring possible symptoms for the safety of oneself as well as the community.

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