What is RT-PCR : Real-time Reverse Transcription Polymer Chain Reaction Corona Virus Test

Covid 19 is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 when a person is infected the most common symptoms include fever cough and shortness of breath to start a test.

What is RT-PCR : Real-time Polymer Chain Reaction Corona Virus Test

The samples can be collected by a nasopharyngeal swab or an oropharyngeal swab for nasopharyngeal specimen. the swab is inserted in the nostril and gently moved forward into the nasopharynx then it is rotated for a specified period time to collect secretions that contain the virus once the swabbing is applied the swab is placed immediately into sterile tube containing a viral transport medium.

The standard method of coronavirus testing is polymerase chain reaction PCR which is a method that used widely in molecular biology to make millions to billions of copies of a specific DNA fragment rapidly coronaviruses contain an extraordinarily long single-stranded RNA genom to detect these viruses with PCR RNA molecules must be converted into their complimentary DNA sequences by reverse transcriptase then the newly synthesized DNA can be amplified by standard pcr procedures.

This approach is universally known as rt-pcr to perform this method basically viral RNA should be extracted several RNA purification kits are available for convenient fast and effective isolation to extract the viral RNA by using commercial kit the sample is first added into a micro centrifuge tube then it is mixed with Elissa's buffer this buffer is highly denaturing and as usually consists of phenol and wanna teen ISO thiocyanate also RNA sin hitters are usually present in the licious buffer to ensure isolation of intact viral  theRNA.

Once licious buffer is added the tube is mixed by pulse vortexing and incubated at room temperature then the virus is lysed under the highly denaturing conditions provided by the licious buffer. once the sample is lysed a purification procedure is carried out by using a spin column the sample is loaded onto the spin column then a centrifugation is performed this procedure is a solid phase extraction method in which the stationary phase consists of a silica matrix under optimal salt and pH conditions RNA molecules are buying to the silica gel membrane and at the same time protein and other contaminants are not retained after centrifugation.

The spin column is placed into a clean collection tube and the filtrate is discarded then a wash buffer is added the column is put in a centrifuge again forcing the wash buffer through the membrane this removes any remaining impurities from the membrane leaving only the RNA bound to the silica gel.

What is RT-PCR : Real-time Polymer Chain Reaction Corona Virus Test

Once the sample is washed the column is placed in a clean micro centrifuge tube and an Aleutian buffer is added then a centrifugation is carried out forcing the elution buffer through the membrane the elution buffer removes the viral RNA from the spin column and a purified RNA which is free of protein inhibitors and other contaminants is obtained after the extraction of the viral RNA.

The next step is the preparation of the reaction mixture for PCR amplification in this step a master mix is used which is a pre-mixed concentrated solution that consists of buffer reverse transcriptase enzyme nucleotides forward primer reverse primer Tackman probe and DNA polymerase finally to complete this reaction mixture the RNA template is added the tube is mixed by pulse vortexing then the reaction mixture is loaded into a pcr plate which generally contains 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 corona virus 2019 by the amplification of target sequences in the RDR P gene the e gene in the end gene the choice of the target gene depends on the primers and the probe sequences the 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 genom reverse transcriptase then adds DNA nucleotides onto the three 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 degrees Celsius and it is used for denaturation of the double-stranded DNA template in the next step the reaction temperature is lowered to 58 degrees Celsius allowing a kneeling of the forward primer to its complimentary 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 five prime to three prime 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.

The reaction temperature is lowered allowing a kneeling of the primers to each of the single-stranded DNA templates and annealing of the Tackman probe to its complementary part of the target DNA Hackman probe consists of a flaw refer covalently attached to the five prime end of the oligonucleotide probe the fluorescence is emitted by the flora fur when it is excited by the cyclers light source also this probe consists of a quencher at the three prime end the close proximity of the reporter to the quencher prevents detection of its fluorescence in the extension step DNA polymer synthesis new strands when the polymerase reaches attackman probe it's endogenous five prime 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.

It is focused into the center of each well then fluorescent light emitted from the wells reflects off the mirror passes through an emission filter and is detected by the CCD camera in each PCR cycle light from excited flora fur can be detected by the CCD camera 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.
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