Coronavirus Test: Real time RT-PCR – Animation video

I make animations in biology with PowerPoint, this animation video is about the standard coronavirus test, real time RT-PCR method, which is a laboratory technique combining reverse transcription of RNA into complementary DNA, and amplification of specific DNA targets using polymerase chain reaction (PCR).

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Transcript

COVID-19 is an infectious disease caused by severe acute respiratory syndrome coronavirus 2When a person is infected, the most common symptoms include fever, cough, and shortness of breathTo start a testThe samples can be collected by a nasopharyngeal swab or an oropharyngeal swab. For Nasopharyngeal specimenthe swab is inserted in the nostril and gently moved forward into the nasopharynxthen it is rotated for a specified period time to collect secretions that contain the virusOnce the swabbing is applied, the swab is placed immediately into sterile tube containing a viral transport mediumThe standard method of coronavirus testing is polymerase chain reaction, PCRWhich is a method that used widely in molecular biology to make millions to billions of copies of a specific DNA fragment rapidlyCoronaviruses contain an extraordinarily long single-stranded RNA genomeTo detect these viruses with PCR, RNA molecules must be converted into their complementary DNA sequences by reverse transcriptaseThen the newly synthesized DNA can be amplified by standard PCR proceduresThis approach is universally known as RT-PCRTo perform this method, basically viral RNA should be extractedSeveral RNA purification kits are available for convenient, fast and effective isolationTo extract the viral RNA by using commercial kitthe sample is first added into a microcentrifuge tube. Then it's mixed with a lysis bufferThis buffer is highly denaturing and is usually consists of phenol, and guanidine isothiocyanateAlso, RNase inhibitors are usually present in the lysis buffer to ensure isolation of intact viral RNAOnce the lysis buffer is added, the tube is mixed by pulse-vortexing, and incubated at room temperatureThen the virus is lysed under the highly denaturing conditions provided by the lysis bufferOnce the sample is lysed, a purification procedure is carried out by using a Spin columnthe sample is loaded onto the spin columnthen a centrifugation is performedThis procedure is a solid phase extraction method, in which the stationary phase consists of a silica matrixUnder optimal salt and pH conditions, RNA molecules are bind to the silica gel membrane, and at the same timeprotein and other contaminants are not retainedAfter centrifugation, the spin column is placed into a clean collection tube, and the filtrate is discarded. Then a wash buffer is addedThe column is put in a centrifuge again, forcing the wash buffer through the membrane.This removes any remaining impurities from the membraneleaving only the RNA bound to the silica gelOnce the sample is washed, the column is placed in a clean microcentrifuge tube, and an elution buffer is addedThen a centrifugation is carried out, forcing the elution buffer through the membraneThe elution buffer removes the viral RNA from the spin columnAnd a purified RNA, which is free of protein, inhibitors, and other contaminants is obtainedAfter the extraction of the viral RNA, the next step is the preparation of the reaction mixture for PCR amplificationIn this step, a master mix is used which is a premixed concentrated solution, that consists of buffer, Reverse Transcriptase enzymeNucleotides, Forward Primer, Reverse Primer, TaqMan probe, and DNA polymeraseFinally, to complete this reaction mixture, the RNA template is addedThe tube is Mixed by pulse-vortexingthen the reaction mixture is loaded into a PCR plate, which generally contain 96 wellsAllowing the analysis of several samples at the same timeNext, the plate is placed in a PCR machine, which is essentially a thermal cyclerReal-time RT-PCR is used for the detection of the new coronavirus 2019by the amplification of target sequences in the Rdrp gene, the E gene and the N geneThe choice of the target gene depends on the primers and the probe sequencesThe first step in RT-PCR is reverse transcriptionThe first-strand complementary DNA synthesis, is primed with the PCR reverse primerwhich hybridizes to a complementary part of the virus RNA genomeReverse transcriptase then adds DNA nucleotides onto the 3-prime end of the primerSynthesizing DNA complementary of the viral RNAThe temperature and duration of this step depend on the primer, the target RNA and the reverse transcriptase usedNext, an initial denaturation step is applied, causing denaturation of the RNA-DNA hybridsThis step is required for the activation of DNA polymeraseand simultaneously the inactivation of reverse transcriptasePCR consists of a series of thermal cycles, with each cycle consisting of Denaturation, Annealing, and Extension stepsDenaturation step consists of heating the reaction chamber to 95 degree Celsius.And it is used for denaturation of the double-stranded DNA templateIn the next stepthe reaction temperature is lowered to 58 degree Celsiusallowing annealing of the forward primer to its complementary part of the single-stranded DNA templateThe annealing temperature relies directly on length and composition of the primersIn the extension step, the DNA polymerase synthesizes a new DNA strandcomplementary to the DNA template strandby adding free Nucleotides from the reaction mixture that are complementary to the template in the 5' to 3' directionThe temperature at this step depends on the DNA polymerase usedAfter the first cycle, the double-stranded DNA target is obtainedThen, the denaturation of this double-stranded DNA is performedyielding two single-stranded DNA moleculesIn the next step, the reaction temperature is lowered, allowing annealing of the primers to each of the single-stranded DNA templatesand annealing of the Taq-man probe to its complementary part of the target DNATaqMan probe consists of a fluorophore covalently attached to the 5' end of the oligonucleotide probethe fluorescence is emitted by the fluorophore when is excited by the cycler’s light sourceAlso, this probe consists of a quencher at the 3' endThe close proximity of the reporter to the quencher prevents detection of its fluorescenceIn the extension step, DNA polymerase synthesizes new strands. When the polymerase reaches a TaqMan probeits endogenous 5' nuclease activity cleaves the probe, separating the dye from the quencherWith each cycle of PCR, more dye molecules are releasedresulting in an increase in fluorescence intensity proportional to the amount of amplicon synthesizedThis method allows the estimation of the amount of a given sequence present in a sampleThe number of double stranded DNA pieces is doubled in each cycletherefore, PCR can be used to analyze extremely small amounts of sample.For the measurement of the fluorescence signala Tungsten- Halogen lampan Excitation filter, Mirrors, lens, an Emission filterand a Charge-coupled device – CCD camera are usedFiltered light from the lamp is reflected off mirror, passes through a condensing lens, and is focused into the center of each wellthen Fluorescent light emitted from the wells reflects off the mirror, passes through an emission filterand is detected by the CCD cameraIn each PCR cycle, Light from excited fluorophore can be detected by the CCDwhich converts the light that it captures into digital dataThis method is known as real time PCRwhich allows the monitoring of the progress of the PCR reaction as it occurs in real time

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