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RT PCR: Real Time- Reverse Transcription Polymerase Chain reaction Test & Procedure

Raina Rahul Agarwal 4 Sep, 2021 Health & Fitness 2 Comments


What is the RT PCR Test?

RT PCR Test is abbreviated as  Real-Time Reverse Transcription  Polymerase Chain Reaction. It is a laboratory test that combines the conversion of RNA into DNA for the detection of a virus. This is an advanced molecular technology for testing of covid 19.  This technology also uses for detecting other diseases. RT PCR  is a well-established technology for the detection quantification of the different microbial agents from various fields like clinical, veterinary, and food safety.

What is RT PCR test For Covid 19?

  • The nasal Swab of the upper respiratory tract of the RT PCR test is an accurate and reliable test for diagnosing Covid 19. A Positive test means you have Covid 19 and a negative test means u did not have Covid 19. This test detects the presence of the virus SARS CoV-2.
  • Real-time PCR involves the conversion of RNA to cDNA ( Copy DNA) via reverse transcription. It follows several cycles of PCR to amplify and detect the target genes of Virus RNA. They do this because only DNA can be copied- or amplified- which is a key part of the real-time RT- PCR process for detecting viruses. The RT PCR test is the gold standard test for covid 19. It was authorised for use in February 2020.
reverse transcription

Source: Reverse Transcriptase & cDNA overview

What is the testing procedure of covid 19?

Covid 19 test conducts in three phases-

1. Sample Collection: Health workers collect the sample with two nose swabs- one swab for nasopharyngeal and the other for oropharyngeal in a VTM tube ( Viral Transport Medium Tube) which is filled with buffer protein and antibiotic. After that, they close the tube with a screw cap and label it ( Name, Age, & Gender). Samples are stored in the boxes and transfer the boxes in the laboratory for RT PCR testing.

sampling for covid 19


2. RNA Extraction: After receiving samples in the laboratory, lab technicians store samples in a 4-5 degree refrigerator. Then samples are processed for RNA extraction of the virus by using Lysis buffer and Wash Buffer and then elute RNA in the tube. These chemical substances remove substances such as proteins and fats and that extract only the RNA present in the sample. The procedure of RNA extraction depends on the  RNA Extraction kit which we are using to extract RNA of viruses. Eluted RNAs are stored in a -20 degree refrigerator.

3. RT PCR Test: RT PCR is the last phase of the testing of covid 19 which takes approximately 1 and a half hour to complete the cycle to give results. PCR machine is a thermal cycler, the machine cycles through temperatures that heat and cool the mixture to trigger specific chemical reactions that create new, identical copies of the target section of viral DNA. The cycle is repeated and each cycle doubles the previous number: two copies become four, four copies become eight, and so on.

In the RT PCR test, microbiologists use the chemicals and Primer/ Probe to make assay mix. This assay mix is mixed with the samples in a PCR plate and then placed in an RT PCR machine. One of the chemicals present in the assay mix that produces fluorescence light if SARS CoV-2 is present in the sample.

Results: Proper amplification of the S-shaped curve will show the results. Special software is used to conduct RT PCR tests and interpret the results of the samples.

Frequently Asked Questions

Que-1 Who should go for the covid 19 test.?

  • A person who has any symptoms of covid 19 such as fever, headache, cough  & cold, loss of taste and smell, etc. can go for covid 19 test.
  • A person who is suspected in contact with covid positive patients.
  • A person who has traveled from one city to another city, one state to another state, and one country to another country should also go for the covid 19 test.

Que-2 Who should not get RT PCR test?

An individual who has already been positive on Rapid Antigen Test (RAT).

Que-3 What is an antibody test?

An antibody test is a type of blood test to see if you have any antibodies for an infection (yes/no result also knows as a qualitative test or how much antibody (a quantitative test). These tests are also referred to as serological tests.

Que-4 What is the difference between the RT PCR test & antigen Test?

RT PCR Test- This test for the presence of the actual virus’s genetic material. PCR is the most reliable and accurate test for detecting active infection. PCR tests take hours to perform, but some are faster.

Antigen Test- This detects bits of protein on the surface of the virus called antigen. Antigen tests typically take only 15 to 30 minutes. Rapid antigen tests are most accurate when used within a few days of the start of your symptoms, which is when the largest amount of virus is present in your body.

Que-5 Which Covid test is more accurate?

The antigen test is faster but is less sensitive than the PCR test. Because the antigen test is not as accurate as PCR, if an antigen test is negative, a patient would be suggested for an RT PCR test to confirm the negative antigen test result.

Conclusion: RT PCR Test is very sensitive. Accuracy of RT PCR test depends on some factors like sample collection, storage temperature, RNA Extraction, and RT PCR Kit and Good Lab Practice, etc.

Disclaimer: This guide is only for information purposes, and the detection of nucleic acid from  SARS- CoV-2 not for any other viruses and pathogens.


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Raina Rahul Agarwal
A Non-Medical Scientist, BSL-2 Lab, Mirzapur

Raina Rahul Agarwal

A Non-Medical Scientist, BSL-2 Lab, Mirzapur

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