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  • 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

  • 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 genome

  • To detect these viruses with PCR, RNA molecules must be converted into their complementary 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 microcentrifuge tube. Then it's mixed with a lysis buffer

  • This buffer is highly denaturing and is usually consists of phenol, and guanidine isothiocyanate

  • Also, RNase inhibitors are usually present in the lysis buffer to ensure isolation of intact viral RNA

  • Once the lysis 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 lysis 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 bind 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

  • Once the sample is washed, the column is placed in a clean microcentrifuge tube, and an elution 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 premixed concentrated solution, that consists of buffer, Reverse Transcriptase enzyme

  • Nucleotides, Forward Primer, Reverse Primer, TaqMan 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 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

  • 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 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, passes 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

COVID-19 is an infectious disease caused by severe acute respiratory syndrome coronavirus 2

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冠狀病毒檢測:實時 RT-PCR - 動畫視頻(Coronavirus Test: Real time RT-PCR - Animation video)

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    1 23 發佈於 2022 年 11 月 23 日
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