Steps of RT-PCR Technique : MED NOW LABS

 

Steps of RT-PCR Technique

As mentioned above, the RT-PCR process is only a little different from that of PCR. Apart from the one addition of reverse transcription in RT-PCR, both techniques are almost the same. So, let’s start the RT-PCR technique from its reverse transcription stage.

Reverse Transcription

A reverse transcriptase enzyme is used to synthesize a cDNA (complementary DNA) strand with nucleotides, and its end product turns out to be mRNA (cDNA hybrid). This process takes place between 40 to 50 degrees Celsius; it usually varies with the properties of the reverse transcriptase enzyme being used. Once the reverse transcription is done, the mRNAs are hydrolyzed, and during a first temperature cycle, the cDNAs are replicated by DNA polymerase.

Denaturation

Here, the combination of components left is heated to a temperature of 94 degrees Celsius for less than half a minute. Along with the denaturation of double-stranded cDNA into single strands, the hydrogen bonds are also broken. These single strands of cDNA then act as templates for producing new DNA strands.

Annealing

This step requires an immediate decrease of temperature to 54-60 degrees for a short period of 20-40 seconds. Next, primers chain up to the DNA sequences, which starts the process of polymerization.

Elongation

In this step, the DNA Taq polymerase enzyme subsequently adds bases to the end of the primer, extending the DNA sequence. It requires the temperature to be somewhere between 72-80 degrees specifically.

Each cycle results in two double-stranded DNA sequences, having one original strand and the other new-made strand in each. As the cycles carry on, these new strands also become templates after every denaturation step. With every cycle, the number of the template doubles, and like this, countless copies of the template are formed. In the end, the same fluorescence technique is also used to detect the presence of SARS-CoV-2 in this process. And that's how the Covid-19 test is considered positive or negative finally. It is also a commonly used technique in Chicago COVID testing.

Isothermal Amplification

Due to the intense spread of Covid-19, saving lives requires quick detection and quick cure of the viral disease. Therefore, places that do not avail proper facilities and expertise for conducting PCR tests need other immediate and amenable options. Isothermal amplification technology is one of such alternatives that are not only manageable in limited settings but has proven its quality comparable to PCR technology too.

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