Everywhere we hear about RT-PCR test these days. Reverse Transcription Polymerase chain reaction (RT PCR) test is required to confirm , if one is Covid-19 positive or negative.
Different types of PCR :
PCR : Polymerase chain reaction is one of widely used molecular biology technique to amplify and detect Deoxy ribo nucleic acid (DNA) and Ribo nucleic acid sequences. This simple technique was invented by Kary Mullis in the year 1985 , where a small amount of DNA can be amplified many folds in quick time. He shared Nobel prize for this discovery in year 1993. The principle of PCR is simple and involves few steps :
- Denaturing of DNA ( separation two strands of DNA by heating)
- Annealing of single DNA strands with small oligonucleotides called primers.
- Extension of primers attached to single DNA strands by DNA polymerase enzyme resulting in two DNA molecules ( remember we had one molecule to begin with)
- Same steps are repeated 25-40 cycles leading to huge amplification of original DNA molecule.
RT-PCR : RT-PCR is similar to PCR but here the starting material is RNA instead of DNA in normal PCR reaction. Since RNA is used as starting material, an extra step is required to convert RNA to DNA. This is accomplished by adding Reverse Transcriptase (RT) enzyme to form cDNA or complimentary DNA. This cDNA fromed from RNA can now be used to amplification.
Real Time PCR or qPCR : Quantitative PCR (qPCR) is used to detect, characterize and quantify nucleic acids for numerous applications in real time.
What is Ct value in qPCR ?
In a real time PCR assay a positive reaction is detected by accumulation of a fluorescent signal. The Ct (cycle threshold) is determined by the number of cycles required for the fluorescent signal to cross the threshold ( i.e, exceeds background level ). Ct levels are inversely proportional to the amount of target nucleic acid (RNA of Sars Cov-2 in case of covid-19 infection) in the sample ( i.e, the lower the Ct level the greater the amount of target nucleic acid in the sample and hence possible more viral load which contributes for more RNA presence in samples).
An individual is considered as Covid-19 negative, if Ct value in RT-PCR report is above 35 and anything less than 35 Ct value is considered as positive.
Mostly the DNA is measured in real time by use of fluorescent dyes and the signal emitted by these dyes allow us to calculate Ct value. Generally high Ct value is less viral load , meaning the sample used for RNA extraction had less viral or no viral particles. A low Ct value in RT-PCR report indicates possible high viral load and signifies the sample had lot of RNA to begin with ( due to presence of more virus particles) and hence less cycles were required to cross the threshold.