Covid-19 RT-PCR Test

Covid-19 RT-PCR Test

Ø Covid-19 most common symptoms are-

·         Fever

·         Cough

·         Shortness of breath

Ø  To the started test sample are collected to person Nasopharyngeal Swab or Oropharyngeal Swab.

Ø  The Collected sample swab placed immediately in Viral transport medium.

Ø  The standard method for the Covid-19 test sample is PCR (polymerase chain reaction).

Ø  PCR is technology, generates sufficient quantities of DNA by increasing the number of copies of the specific target region of DNA.

Ø  Coronavirus is contain extra-ordinary single standard RNA genes, to take this single RNA in PCR must be converted complementary strands.

Ø  Then the newly synthesize DNA can be amplified in PCR that called RT-PCR (Reverse transcriptase polymerase chain reaction).

To Performed RT-PCR:

Ø  The collected sample in viral transport medium transfer to the micro-centrifuge tube with the help of the micropippet.

Ø  Then the mixed Lysis buffer in the centrifuge tube. This buffer is highly denaturing nad usually phenol and guanidine isothiocyanate.

Ø  Once the buffer added mixed with vortaxt machine.

Ø  Incubate at room temperature.

Ø  Then the virus is highly denaturing conditions by Lysis buffer.

Ø  Once the virus is denaturing using the spin columns, sample are loaded in the spin columns and centrifuge.

Ø  Which the stationary phase columns are made with silica matrix and salt or pH condition.

Ø  In this process RNA binds to the silica gel and protein or other contaminant remove to the columns.

Ø  After the centrifuge spin columns are placed into clean columns and filtred are dicarded.

Ø  Then the wash buffer was added and the columns are placed in centrifugation.

Ø  Wash buffer can remove the any other impurity to columns that RNA only can bind to the silica gel.

Ø  Again columns placed clean microcentrifuge tube and Elution buffer added.

Ø  Placed centrifugation machine and centrifuge.

Ø  Elution buffer can remove the RNA in the columns.

Next steps Reaction mixture preparation:

Ø  In this steps the master mix the sample RNA.

Ø  In the master mixer contain Reverse transcriptase(RT), dNTPs, Reverse primer, forward primer, TaqMan probe and DNA polymerase.

Ø  Finally, the complete the reaction mixture sample are added into the master mix.

Ø  The tube is mixed with the vortex machine properly.

Ø  After the mixture loaded in PCR plate.

Ø  Next the plate is placed PCR machine which is thermal circular.

Ø  Then the PCR is detected the Covid-19 RNA by amplification of the target sequences of target sequences RdRP gene, E gene and N gene.

Ø  Choose the gene depends on the primers of the probe sequences.

The first step RT-PCR is reverse transcription:

Ø Complementary DNA synthesis prime with PCR reverse primer which hybridization with complementary Virus RNA and added to nucleotide to 5’ to 3’ ends.

Ø  Temperature depends on the primers target RNA and Reverse RNA are used.

Initial Denaturation:

Ø  The denaturation step is requires 95⁰C temperature for denaturing cDNA strands.

Ø  They can remove the double stranded cDNA to single stranded.

Annealing:

Ø  Allowing to temperature at 58⁰C.

Ø  In this process attached to the forward primer to complementary part of the strands.

Extension:

Ø  The temperature requires 58⁰C.

Ø  DNA polymerase enzymes synthesis to adding nucleotide to the complementary strands to 5’ to 3’ ends.

Ø  After the first cycle again denaturation process to the DNA strands.

Ø  The next step annealing the temp. lower to the 58⁰C.

Ø  The allowing the synthesis of the single stranded DNA strands.

Ø  Taq. Probe attached to the target DNA sequence Genes.

Ø  Extention to new synthesized of the new DNA.

Ø  After the many cycles are run to PCR Machine.

Ø  Number of double stranded DNA sequence is allowing after the many cycle of PCR.

Ø  The result are show to the help of the Tungsten Halogen lamp in the RT-PCR detections.

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