Real-Time RT-PCR (Reverse Transcriptase Polymerase Chain Reaction) is the gold standard test for detecting cases of COVID-19 caused by the SARS-CoV 2 virus. The test requires a specialized laboratory setup with specific biosafety and biosecurity precautions to be followed. The average time taken is around 4-6 hours from receipt of the sample to getting the result. The advantage of this platform lies in its accuracy of detection Sensitivity of the test may vary from 80-95% and specificity is usually above 95 %.
The procedure of the test
The COVID-19 virus generally gathers in the throat or inside the nose of a person. The RT-PCR Test diagnosis process starts with sample collection of Oropharyngeal and Nasopharyngeal swabs in viral transport media maintained at 2-8 ᵒC.
The cells and nucleus are lysed by treating them with different chemical solutions. Finally, the sample only contains DNA and RNA. It is a combination of the residual genetic material of the individual and the RNA of the virus. The RNA is then converted to DNA using a specific enzyme via reverse transcription. Short fragments of DNA are also added to create a mixture.
If there is a virus in the sample, then the short fragments of the DNA accord to the target divisions of the viral DNA.
The mixture is then put into an RT–PCR machine. The RT-PCR machine heats and cools the mixture by cycling it through temperatures to ignite specific chemical reactions. Through this process, the target sections (RdRp gene , E gene, N gene) of viral DNA get their new identical copies.
The RT-PCR COVID-19 Test machine carries out repeated cycles with the sample. This helps in copying the target sections of the viral DNA. At the end of each cycle, the number of copies of the viral DNA gets doubled. As a result, new copies of the viral DNA sections from each strand of the virus are generated by the end of the process.
Once the new copies of the viral DNA are formed, the marker labels then attach to the DNA strands. The RT-PCR machine is connected to a computer that records the amount of fluorescent dye released after each step. It displays the result on the screen at the same time. At a certain point, when the amount of fluorescence crosses a particular limit, this confirms the presence of the virus.
The fewer the cycles to confirm the virus, the more severe is the condition. Therefore, when reading the RT-PCR report you need to check whether the CT value is below or above 35. A lower value is positive for COVID-19 and a higher value is negative for the disease. One should take proper care of themselves as per the guidance of the physician to prevent the spreading of the virus.
The RT-PCR test result can be either positive or negative. A positive RT-PCR test means that you are likely to be infected with COVID-19 virus.
False-positive results are rare but can occur through mislabelling, transcription errors, sample contamination and amplification of nonspecific products. Results with high CT values close to the maximum number of cycles (35-40) need to be interpreted more carefully. Detection of virus in asymptomatic individuals may represent asymptomatic or pre-symptomatic infection or detection of RNA following clinical recovery (the median duration of detection in respiratory samples is around 18 days (13-29 days).
False-negative results can occur if samples are collected inadequately, through mislabelling or if the wrong site is sampled (for example lower respiratory tract samples recommended if pneumonia is present).
In view of the specialized laboratory requirements, this test cannot be performed at every district level lab which do not have molecular virology facilities. However, wherever available, it is advised to use real-time RT-PCR as the frontline test for the diagnosis of SARS-CoV-2.
