Coronavirus Test: Real time RT-PCR - Animation video

Summary

The video script explains the process of testing for COVID-19 using a method called RT-PCR, which stands for Reverse Transcription Polymerase Chain Reaction. It begins with the collection of samples through a nasopharyngeal or oropharyngeal swab. The swab is then placed into a sterile tube with a viral transport medium. The RT-PCR technique involves converting the virus's RNA into DNA, which is then amplified to create many copies for detection. The process includes RNA extraction, purification using a Spin column, and preparation of a reaction mixture for PCR. The PCR machine amplifies and detects the virus's genetic material by targeting specific genes. Real-time monitoring of the PCR reaction allows for the estimation of the virus's quantity in the sample. The method's sensitivity makes it suitable for detecting even minute amounts of the virus, crucial for diagnosing COVID-19.

Takeaways

• 🦠 COVID-19 is caused by the SARS-CoV-2 virus and presents with symptoms like fever, cough, and shortness of breath.
• 🌡️ Testing for COVID-19 involves collecting samples via nasopharyngeal or oropharyngeal swabs.
• 🧪 The standard testing method is RT-PCR, which amplifies the virus's RNA into DNA and then makes multiple copies for detection.
• 🧬 The conversion of viral RNA to DNA is done using reverse transcriptase, a key step in RT-PCR.
• 🧴 A lysis buffer is used to break down the virus and extract the RNA, which is then purified using a Spin column method.
• 🧬 RNA purification ensures that the viral RNA is free from proteins, inhibitors, and other contaminants.
• 🧪 The PCR master mix includes essential components like buffer, enzymes, primers, probe, and DNA polymerase.
• 🔬 Real-time RT-PCR detects the virus by amplifying target sequences in specific genes like Rdrp, E, and N.
• 🔥 The PCR process involves thermal cycles of denaturation, annealing, and extension to create new DNA strands.
• 🔬 TaqMan probe technology is used in real-time PCR, where fluorescence indicates the presence and quantity of the target DNA.
• 📈 The increase in fluorescence intensity with each PCR cycle allows for the real-time monitoring and quantification of the virus's genetic material.
• 🛠️ The PCR machine uses a combination of a light source, filters, and a CCD camera to detect and convert fluorescence into digital data for analysis.

Q & A

What is COVID-19 caused by?

COVID-19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

What are the most common symptoms of a person infected with COVID-19?

The most common symptoms of COVID-19 include fever, cough, and shortness of breath.

How are samples collected for COVID-19 testing?

Samples for COVID-19 testing can be collected by a nasopharyngeal swab or an oropharyngeal swab.

What is the standard method used for coronavirus testing?

The standard method for coronavirus testing is the polymerase chain reaction (PCR).

How is the viral RNA extracted from the sample?

The viral RNA is extracted by adding the sample to a microcentrifuge tube with a lysis buffer, followed by a solid phase extraction method using a Spin column.

What is the purpose of the lysis buffer in the RNA extraction process?

The lysis buffer is used to break down the virus and release the RNA, while also containing RNase inhibitors to protect the RNA from degradation.

What is the role of a Spin column in the RNA purification process?

The Spin column is used to bind the RNA to a silica gel membrane while allowing proteins and other contaminants to be washed away.

What is the name of the approach used to amplify the viral RNA for PCR?

The approach used to amplify the viral RNA for PCR is known as reverse transcription polymerase chain reaction (RT-PCR).

What is the purpose of the master mix in the PCR amplification step?

The master mix is a premixed concentrated solution containing all the necessary components for PCR, such as buffer, enzymes, primers, and probes, to facilitate the amplification of the target DNA.

How does the TaqMan probe work in real-time RT-PCR?

The TaqMan probe is an oligonucleotide probe with a fluorophore and a quencher. When the probe is cleaved by the DNA polymerase during PCR, the fluorophore is separated from the quencher, emitting a fluorescence signal that increases with each PCR cycle, allowing for the real-time monitoring of amplification.

What is the significance of the increase in fluorescence intensity during real-time RT-PCR?

The increase in fluorescence intensity is proportional to the amount of amplicon synthesized, allowing for the estimation of the starting quantity of the target sequence in the sample.

How does real-time PCR allow for the monitoring of the PCR reaction?

Real-time PCR uses a charge-coupled device (CCD) camera to detect the fluorescent light emitted from the wells during each cycle, converting it into digital data that can be used to monitor the progress of the PCR reaction as it occurs.

Outlines

00:00

🧫 COVID-19 Testing and RT-PCR Methodology

This paragraph explains the process of COVID-19 testing using RT-PCR. It begins with the collection of samples through nasopharyngeal or oropharyngeal swabs. The swab is inserted into the nostril and rotated to collect secretions containing the virus, which are then placed into a sterile tube with a viral transport medium. The standard testing method, RT-PCR, involves converting the virus's RNA into complementary DNA using reverse transcriptase, followed by amplification. The process includes RNA extraction using a commercial kit, which involves mixing the sample with a lysis buffer to break down the virus and isolate the RNA. The RNA is then purified using a Spin column method, which binds the RNA to a silica matrix while discarding other contaminants. The purified RNA is used in a master mix for PCR amplification, which includes various components like buffer, enzymes, and primers. The RT-PCR process involves reverse transcription, denaturation, and the amplification of target sequences in specific genes of the coronavirus.

05:06

🔬 PCR Amplification and Real-Time Detection

The second paragraph delves into the specifics of the PCR process, which includes a series of thermal cycles consisting of denaturation, annealing, and extension. Denaturation separates the DNA strands, annealing allows primers to bind to the DNA template, and extension synthesizes new DNA strands. The TaqMan probe, attached with a fluorophore and a quencher, is used to monitor the PCR progress. As the DNA polymerase cleaves the probe during extension, the fluorescence intensity increases, which is proportional to the amount of amplicon produced. This real-time PCR allows for the estimation of the target sequence's quantity in the sample. The fluorescence signal is measured using a series of optical components, and the emitted light is captured by a CCD camera, which converts it into digital data. This enables monitoring the PCR reaction in real time, facilitating the detection and quantification of the coronavirus from the sample.

Keywords

💡COVID-19

COVID-19 is an infectious disease caused by the novel coronavirus known as SARS-CoV-2. It is characterized by symptoms such as fever, cough, and shortness of breath. In the context of the video, understanding COVID-19 is crucial as it is the subject of the diagnostic process being described. The video script discusses the methods used to detect the virus causing this disease.

💡Nasopharyngeal swab

A nasopharyngeal swab is a procedure where a拭子 (swab) is inserted into the nostril and moved forward into the nasopharynx to collect secretions that may contain the virus. This is a key method for collecting samples for COVID-19 testing, as mentioned in the script when describing the sample collection process.

💡Polymerase Chain Reaction (PCR)

PCR is a widely used method in molecular biology that allows for the rapid amplification of specific DNA fragments, creating millions to billions of copies. In the context of the video, PCR is the standard method for testing the presence of coronavirus. It is used to amplify the viral RNA that has been converted to DNA, enabling the detection of the virus.

💡Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

RT-PCR is a technique that combines reverse transcription, which converts RNA to DNA, with PCR to amplify the DNA. This method is essential for detecting coronaviruses, which have an RNA genome. The script explains that the RNA from the virus must first be converted to DNA before it can be amplified using standard PCR procedures.

💡RNA purification

RNA purification is the process of isolating RNA from a sample. This is a critical step in the RT-PCR process as it ensures that the RNA is free from proteins and other contaminants that could interfere with the test. The script describes the use of commercial kits and a Spin column for this purpose.

💡Lysis buffer

A lysis buffer is a highly denaturing solution used to break open the virus and release its RNA. It typically contains substances like phenol and guanidine isothiocyanate. In the script, it is mentioned as part of the RNA purification process where the sample is mixed with the lysis buffer to lyse the virus.

💡Spin column

A Spin column is a device used in the purification process to separate components of a sample through centrifugation. The script describes its use in binding RNA to a silica matrix while allowing other contaminants to be washed away, resulting in purified viral RNA.

💡Master mix

A master mix is a premixed concentrated solution used in PCR that contains all the necessary components for the reaction, except for the RNA template. It includes buffer, reverse transcriptase enzyme, nucleotides, primers, and DNA polymerase. The script explains that this mix is used to prepare the reaction mixture for PCR amplification.

💡Real-time RT-PCR

Real-time RT-PCR is a variation of RT-PCR that allows for the monitoring of the PCR reaction in real-time. It is used to detect the presence of the new coronavirus by amplifying target sequences in specific genes. The script details how this method enables the estimation of the amount of a given sequence in a sample and is central to the diagnostic process for COVID-19.

💡TaqMan probe

A TaqMan probe is a type of probe used in real-time PCR that has a fluorophore and a quencher attached to it. When the probe is intact, the fluorescence is not detected. However, when the probe is cleaved by the DNA polymerase during the extension step, the fluorophore is separated from the quencher, and the fluorescence is emitted. This allows for the detection and quantification of the target DNA. The script describes its role in the PCR process.

💡Thermal cycler

A thermal cycler is a machine that automates the PCR process by controlling the temperature changes necessary for the various steps of the PCR, such as denaturation, annealing, and extension. The script mentions the PCR machine, which is essentially a thermal cycler, as the device used to carry out the PCR cycles.

💡Fluorescence

Fluorescence is the emission of light by a substance (the fluorophore) when it is excited by a light source. In the context of the video, fluorescence is used to detect the amplification of the target DNA during real-time RT-PCR. The increase in fluorescence intensity is proportional to the amount of amplicon synthesized, allowing for the monitoring of the PCR progress.

Highlights

COVID-19 is caused by the SARS-CoV-2 virus, with common symptoms like fever, cough, and shortness of breath.

Testing for COVID-19 involves collecting samples via nasopharyngeal or oropharyngeal swabs.

Nasopharyngeal swab involves inserting the swab into the nostril and rotating it to collect secretions.

The swab is placed in a sterile tube with a viral transport medium after collection.

Polymerase Chain Reaction (PCR) is the standard method for detecting coronaviruses.

Coronaviruses have a long single-stranded RNA genome that must be converted to DNA for PCR testing.

RT-PCR is used to amplify the DNA converted from the viral RNA for detection.

Viral RNA extraction is facilitated by commercial kits using a lysis buffer.

RNA purification involves a Spin column and solid phase extraction method.

Purified RNA is obtained by washing the Spin column and eluting the RNA with a buffer.

PCR amplification requires a master mix with components like buffer, enzymes, primers, and probe.

Real-time RT-PCR detects the new coronavirus by amplifying target sequences in specific genes.

The RT-PCR process includes reverse transcription, denaturation, annealing, and extension steps.

TaqMan probe technology is used in real-time PCR for detecting the increase in fluorescence with each cycle.

PCR allows for the analysis of extremely small sample amounts by doubling the target DNA in each cycle.

Real-time PCR monitoring uses a combination of a light source, filters, and a CCD camera to detect fluorescence.

The increase in fluorescence intensity is proportional to the amount of amplicon synthesized, enabling sequence quantity estimation.

The RT-PCR process is monitored in real-time, providing immediate results as the reaction progresses.

Transcripts

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