Corona virus (COVID – 19) Test Real Time RT-PCR

 COVID – 19 Infectious disease caused by sever acute respiratory syndrome corona virus 2. When a person is infected by this virus , the most common symptoms include Fever, cough, and shortness of  Breath. RT-PCR is used in testing of corona virus here we talk about only process held in the RT-PCR machine. Reaction mixture*, the RNA template is added, The tube is Mixed by pulse-vortexing then the reaction mixture is loaded into a PCR plate,

   PCR –Plate

                                     Real time RT-PCR machine                        

  which generally contain 96 wells Allowing the analysis of several samples at the same time Next, the plate is placed in a PCR machine, which is essentially a thermal cycler Real-time RT-PCR is used for the detection of the new coronavirus 2019 by the amplification of target sequences in the Rdrp gene,  the E gene and the N gene The choice of the target gene depends on the primers and the probe sequences. first step in RT-PCR is reverse transcription The first-strand complementary DNA synthesis, is primed with the PCR reverse primer which hybridizes to a complementary part of the virus RNA genome Reverse transcriptase then adds DNA nucleotides onto the 3-prime end of the primer Synthesizing DNA complementary of the viral RNA The temperature and duration of this step depend on the primer, the target RNA and the reverse transcriptase used Next, an initial Denaturation step is applied, causing denaturation of the RNA-DNA hybrids This step is required for the activation of DNA polymerase and simultaneously the inactivation of reverse transcriptase .PCR consists of a series of thermal cycles, with each cycle consisting of Denaturation, Annealing, and Extension steps Denaturation step consists of heating the reaction chamber to 95 degree Celsius. And it is used for denaturation of the double-stranded DNA template

In the next step:The reaction temperature is lowered to 58 degree Celsius allowing annealing of the forward primer to its complementary part of the single-stranded DNA template The annealing temperature relies directly on length and composition of the primers In the extension step, the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding free Nucleotides from the reaction mixture that are complementary to the template in the 5' to 3' direction The temperature at this step depends on the DNA polymerase used After the first cycle, the double-stranded DNA target is obtained Then, the denaturation of this double-stranded DNA is performed yielding two single-stranded DNA molecules In the next step, the reaction temperature is lowered, allowing annealing of the primers to each of the single-stranded DNA templates and annealing of the Taq-man probe to its complementary part of the target DNA TaqMan probe consists of a fluorophore covalently attached to the 5' end of the oligonucleotide probe the fluorescence is emitted by the fluorophore when is excited by the cycler’s light source Also, this probe consists of a quencher at the 3' end The close proximity of the reporter to the quencher prevents detection of its fluorescence In the extension step, DNA polymerase synthesizes new strands. When the polymerase reaches a TaqMan probe its endogenous 5' nuclease activity cleaves the probe, separating the dye from the quencher With each cycle of PCR, more dye molecules are released resulting in an increase in fluorescence intensity proportional to the amount of amplicon synthesized This method allows the estimation of the amount of a given sequence present in a sample The number of double stranded DNA pieces is doubled in each cycle therefore, PCR can be used to analyze extremely small amounts of sample. For the measurement of the fluorescence signal a Tungsten- Halogen lamp an Excitation filter, Mirrors, lens, an Emission filter and a Charge-coupled device - CCD camera are used Filtered light from the lamp is reflected off mirror, passes through a condensing lens, and is focused into the center of each well then Fluorescent light emitted from the wells reflects off the mirror, 

through an emission filter and is detected by the CCD camera In each PCR cycle, Light from excited fluorophore can be detected by the CCD which converts the light that it captures into digital data This method is known as real time PCR which allows the monitoring of the progress of the PCR reaction as it occurs in real time

Reaction mixture * : master mix (premixed concentrated solution, that consists of buffer, Reverse Transcriptase enzyme Nucleotides, Forward Primer, Reverse Primer, TaqMan probe, and DNA polymerase) & viral  RNA template ( sample )

-Ramnath p