What is RT-PCR Test and how it works exactly?

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today's scenario when the world is struggling and fighting against the novel coronavirus every one of you must be aware of RT-PCR tests. RT-PCR or the real-time polymerase chain reaction is the most reliable test for the detection of covid19 infection to date. if you are a college student or a researcher, you should definitely not miss the video because in today's video I have tried to cover as much information regarding PCR based on the resources and my personal experience. In this video, you will learn what anRT-PCR is. how it is different from the conventional PCR two of the most popular types of RT-PCRsi.e., the intercalating dye-based and probe-based and their working principles. what are the requirements of performing RT-PCR tests? taking an example of covid19. how the probes and the primers are designed? how the RT-PCR test actually works interpretation of the RT-PCR results. and at last some of the major advantages and applications of RT-PCR. 

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so let's begin with what RT-PCR is? RT-PCR is short for a real-time polymerase chain reaction. itis a PCR variant in which one can detect and quantify the expression of a target gene based on the fluorescence produced upon template amplification. in RT-PCR the mRNA is the initial template that gets reversed transcribed to cDNA.this DNA undergoes several rounds of PCR amplification which leads to an increase in the fluorescence signal detected by the machine confuses RT-PCR with the reverse transcript is PCR because it involves the reverse transcription of mRNA to cDNA in its initial steps. now let's see how RT-PCR is different from a normal or a conventional PCR. in conventional PCR you can amplify a gene DNA locus which is later detected on a gross gel or a PAGE. now it has two major problems one that with the conventional PCR precise quantification of gene expression is not possible

and secondly, the DNA template required is significantly more as EtBr is less sensitive than the fluorescence dye real-time PCR on the other hand precisely detects and quantifies the expression of the target gene in real-time manner after each PCR cycle as it is fluorescence-based it is more sensitive in detecting minor differences in the gene expression machine that performs real-time PCR is equipped with the fluorescence detection and quantification system fluorescence-based detections makes it 1000 times more sensitive than conventional PCR also the template that is required is in few picograms based on the chemistry used two major types of RT-PCR exists the first one is the intercalating fluorescent dye-based in this chemistry as the cDNA gets amplified in consecutive PCR cycles more and more double-stranded DNA products become available for the intercalating dye like SYBRgreen, shown here with the green star to bind and show fluorescence once sufficient fluorescence is produced it is detected by the instrument and reflects the expression of the target gene.

this type of PCR is very convenient cheaper and popular in most laboratories but at certain times, it faces the problem of non-specific amplification which still produces fluorescence signal due to its double-stranded nature giving false results melt curve analysis program gives some idea about non-specific amplicons. the second rt-PCR type is a fluorescent probe based which produces much more specific and reliable results this type of RT-PCR variant is most popular for COVID 19 detection utilizes stickman probes containing fluorophore and a quencher molecule in its close proximity during PCR amplification tacman polymerases make a new copy of complementary DNA targets it reaches towards the probe, it cleaves and frees the fluorophore from the quencher with its 5'-3' exonuclease activity during successive PCR cycles more and more probes releases fluoro phone producing detectable fluorescence signal probe-based detection increases the accuracy of detection of multiple probes labeled with the different fluorophores.

 molecules can be multiplexed together making it easier to perform multiplex PCR for multiple targets however the probe designing makes this technique much more complex from this point onwards I will be focusing on the probe-based rt-PCR which is used for the covid19 detection now let's talk about the requirements for rt-PCR the sample of the individual to be tested as the covid-19 virus infects the upper respiratory tract of the individuals most commonly nasopharyngeal or the oropharyngeal swabs are used to perform the testing the COVID 19 suspect later on the RNA is extracted from these samples the second requirement is the RT-PCR kit contains a mixture of components such as the primers and the probes specific for COVID 19 virus the polymerase dNTPs etc.

 this mix the RNA sample is added usually rt PCR tests are performed in 96well plates to carry out analysis of multiple samples and save time thermocycler so this is the instrument that performed the PCR by varying the temperature and comes with a computer for the analysis of resource how the primer and the probe are designed this one is the most important factor that determines the accuracy of the RT-PCR results poor designing leads to false results and if the sample is of COVID 19suspect this part cannot be overlooked the primers are the oligonucleotide sequences of 18 to 24 pair long and the probes are usually shorter of 8 to 12 base pair for COVID 19 detection two or more primer pairs and probes specific for the covid-19nucleocast regions are designed using software like primer3.

 human RNase P is also taken as a control to check PCR success their melting temperature self-complementarity and loop formation ability is appropriately chosen for successful PCR amplification how RT-PCR actually works the RNA extracted from the suspected person is added to a PCR tube or 96 well plates containing the RT-PCR master mix is then put in a thermocycler wherein a PCR program is set which comprises of denaturationannealing and extension at appropriate temperatures for amplification of target genes during denaturation the double-stranded DNA is separated and as the temperature is lowered primers and probes anneal to the template when the polymerase starts elongating a new copy of DNA.

 encounters the probe and with its 5 -3' exonuclease activity cleaves the probe releasing the fluorophore from the close proximity of the quencher and leads to the emission of fluorescence after successive cycles more and more floor gets released which is detected by the instrument in a real-time manner now how to interpret the results after the amplification is over the results are shown in the form of values or the threshold cycle number ct reflects the quantity of target mRNA in the sample lower the ct value higher the copy number of the target often ct values are normalized with internal control or the housekeeping gene improve the reliability of the results the tests are performed in replicates and their statistical significance is determined based on the standard curves absolute quantification is also possible relative expression among various samples may also be studied in case of covert 19 low ct value represents high infection if ct value is over 35 it means very.

low or no viral infection is present now talking about the advantages and applications of rtpcras the RT-PCR results are available in two to three hours hence it is a very rapid method of detection and quantification of gene expression fluorescence-based detections makes it a very sensitive technique a man probe-based rt-PCRs are much more reliable and yields reproducible results different fluorophore tagged probes maybe designed hence multiple target genes can be studied simultaneously in a single reaction rtpcrs find their application in detection of various diseases the best example in today's scenario is the loved 19. rdpcrs are also used to check the relative expression of the target gene in a different type of samples for example normal versus cancerous cells once a standard curve is made actual quantity of target gene expressed may also be determined rt PCRs can easily detect the microbial load in samples such as contaminated water soil samples etc. 

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