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COVID-19 is an infectious disease caused by Severe Acute Respiratory Syndrome Coronavirus Type 2. When a person is infected, the most common symptoms include fever, cough, and shortness of breath.

news_001The samples used for testing can be collected by nasopharyngeal swabs or oropharyngeal swabs.

news_002What is PCR?

The standard method of coronavirus detection is polymerase chain reaction, PCR. This is a method widely used in molecular biology. It can quickly copy millions to billions of specific DNA fragments.

news_003The new coronavirus contains a very long single-stranded RNA genome. In order to detect these viruses by PCR, RNA molecules must be converted into their complementary DNA sequences by reverse transcriptase, and then the newly synthesized DNA can be amplified by standard PCR procedures, which is commonly known as RT-PCR.

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RT-PCR process

RNA extraction

To perform this method, viral RNA should basically be extracted. A variety of RNA purification kits can be used for convenient, fast and effective separation.

To extract viral RNA using a commercial kit, first add the sample to a microcentrifuge tube and then mix it with the lysis buffer. This buffer is highly denatured and usually consists of phenol and guanidine isothiocyanate. In addition, RNase inhibitors are usually present in the lysis buffer to ensure isolation of intact viral RNA.

news_005After adding the lysis buffer, vortex the mixing tube by pulse and incubate at room temperature. The virus is then lysed under highly denaturing conditions provided by the lysis buffer.

news_006After the sample is lysed, a centrifuge tube is used for the purification procedure. The sample is loaded into the centrifuge tube and then centrifuged.

news_007This procedure is a solid phase extraction method in which the stationary phase consists of a silica gel matrix.

news_008Under optimal salt and pH conditions, RNA molecules bind to the silica membrane.

news_009At the same time, protein and other contaminants are removed.

news_010After centrifugation, put the centrifuge tube into a clean collection tube, discard the filtrate, and then add washing buffer.

news_011Place the tube in the centrifuge again to force the wash buffer through the membrane. This will remove all remaining impurities from the membrane, leaving only the RNA bound to the silica gel.

news_012After the sample is washed, put the tube into a clean microcentrifuge tube and add the elution buffer.

news_013It is then centrifuged to force the elution buffer through the membrane. The elution buffer removes viral RNA from the spin column and obtains purified RNA free of proteins, inhibitors, and other contaminants.

news_014STEP 2

Mixed concentrate

After extracting the viral RNA, the next step is to prepare the reaction mixture for PCR amplification. In this step, concentrate is used. This concentrated solution is a premixed concentrated solution consisting of a premix, reverse transcriptase, nucleotides, forward primer, reverse primer, TaqMan probe and DNA polymerase.

news_015Finally, to complete this reaction mixture, the RNA template is added. The tubes are mixed by pulse vortexing, and then the reaction mixture is loaded into the PCR plate. The PCR plate usually contains 96 wells and can analyze multiple samples at the same time.

news_016STEP 3

PCR amplification

Next, place the plate in the PCR machine, which is essentially a thermal cycler.

news_017Real-time RT-PCR is used to detect the 2019 novel coronavirus by amplifying the target sequence in the RdrRP gene, E gene and N gene. The choice of target gene depends on the primer and probe sequence.

news_018The first step of RT-PCR is reverse transcription. The first strand of complementary DNA is synthesized, which is initiated by the PCR reverse primer, which binds to the complementary part of the viral RNA genome. Then reverse transcriptase adds DNA nucleotides to the 3′end of the primer to synthesize DNA complementary to the viral RNA. The temperature and duration of this step depend on the primers, target RNA, and reverse transcriptase used.

news_019Next, an initial denaturation step is applied, which results in the denaturation of the RNA-DNA hybrid. This step is necessary to activate the DNA polymerase. At the same time, reverse transcriptase is inactivated.

news_020PCR consists of a series of thermal cycles. Each cycle consists of denaturation, annealing and extension steps.

news_021The denaturation step involves heating the reaction chamber to 95 degrees Celsius and using it for denaturation of the double-stranded DNA template.

news_022In the next step, the reaction temperature is reduced to 58 degrees Celsius, allowing the forward primer to anneal to the complementary part of its single-stranded DNA template. The annealing temperature directly depends on the length and composition of the primer.

news_023In the extension step, the DNA polymerase synthesizes a new DNA strand that is complementary to the DNA template strand. By adding free nuclei complementary to the template in the 5′to 3′direction from the reaction mixture. The temperature of this step depends on the DNA polymerase used.

news_024After the first cycle, a double-stranded DNA target is obtained.

news_025Then, enter the second cycle. The double-stranded DNA is denatured to produce two single-stranded DNA molecules.

news_026In the next step, the reaction temperature is lowered, the primers are annealed to each single-stranded DNA template, and the Taq-man probe is annealed to the complementary part of the target DNA.

news_027The TaqMan probe consists of a fluorophore covalently linked to the 5′end of the oligonucleotide probe. When excited by the light source of the cycler, the fluorophore emits fluorescence. In addition, the probe is composed of a quencher at the 3′end. The proximity of the reporter gene to the quencher prevents the detection of fluorescence.

news_028In the extension step, the DNA polymerase synthesizes a new strand. When the polymerase reaches the TaqMan probe, its endogenous 5′nuclease activity cleaves the probe, separating the dye from the quencher.

news_029With each cycle of PCR, more dye molecules are released, resulting in an increase in fluorescence intensity proportional to the number of amplicons synthesized.

news_030This method allows to estimate the number of a given sequence present in the sample. The number of double-stranded DNA fragments doubles in each cycle. Therefore, PCR can be used to analyze very small samples.

news_031For measuring fluorescent signal, tungsten halogen lamp, excitation filter, reflector, lens, emission filter and charge coupled device-use CCD camera.

STEP 4 Detect

For measuring fluorescent signal, tungsten halogen lamp, excitation filter, reflector, lens, emission filter and charge coupled device-use CCD camera.

news_032The filtered light from the lamp is reflected by the reflector, passes through the condenser lens, and is focused to the center of each hole. Then the fluorescence emitted from the hole is reflected from the mirror, passes through the emission filter, and is detected by the CCD camera. In each PCR cycle, the self-excited fluorophore light can be detected by the CCD.

news_033It converts the captured light into digital data. This method is called real-time PCR, and it allows real-time monitoring of the progress of the PCR reaction.

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Post time: Jul-19-2021