What is RT-PCR? 

Reverse Transcriptase - Polymerase Chain Reaction is a labarotary based technique used for detecting and comparing the level of RNAs and surface proteins in a sample with limited quantity of RNA. 

How is the diagnosis done in RT-PCR test? 

For the diagnosis in RT-PCR test, sample is collected through Nasopharyngeal or oropharyngeal swab or from saliva sample recently. The swab is then maintained in a lysis buffer. The testing of the swab is then done using a RT-PCR machine 

Microbiological mechanism of RT-PCR test 

• Isolation of RNA sample and creating complimentary DNA

SARS-COV-2 is a RNA virus. So, in order to detect an RNA virus, all the RNA is extracted and isolated from the patient. The isloted RNA is difficult to work with in laboratory as it does not undergo PCR. Therefore, single stranded RNA is converted into double stranded DNA with the help of Reverse Transcriptase enzyme.

• Polymerase Chain Reaction

The detection of SARS-CoV-2 is now done with the help of PCR. PCR amplifies and quantifies DNA molecule. PCR contains three steps:

1. Denaturation
2. Annealing
3. Elongation

Each step should be repeated 40 cycles in order to achieve the desired amplification. 

1.Denaturation 

In the first step, all the double stranded molecules are denatured. It means the double strands are separated from each other. 

2.Annealing

A pair of primers, specific to SARS-CoV-2, attach to each separated strand of DNA. These primers only bind to viral c-DNA. 

3.Elongation 

An enzyme Polymerase adds nucleotide to the end of primers. This is done to create two double stranded DNA molecule. 

• Addition of probe and checking the cT value

In real time RT-PCR a probe is added during PCR which gives off fluorescence each time a new DNA is formed. The increase in viral c-DNA can be followed by tracking an increase in flouroscent signal. When level of fluoroscence increases a certain threshold, we can be sure that the signal is significantly above than the background level. 

The cycle threshold (cT) value is the number of cycle required for the flouroscent signal to exceed that specific threshold value. The lower the cT value, the more of RNA was present in the original sample, indicating a higher viral load. 

Conclusion 

In this rising risk of getting infected with SARS-COV-2, it has become extremely important to get ourselves tested again and again. This test is 100% safe and the results are 99% true and trustworthy. Now that you have known the mechanism of this test there should be no fear or doubt while taking it.