How we test for SARS-CoV-2 - RT-PCR (Reverse Transcription PCR)
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- čas přidán 13. 09. 2024
- Hey scientists,
today I will present you a technique which is used for the testing of patients whether they are infected with a certain virus or not. This is for example used in the Covid-19 Corona virus crisis right now and the RNA extracted from patients undergoes exactly this mechanism described here.
This literature here was helpful for me:
www.thermofish...
Video about PCR (Polymerase Chain Reaction):
• How PCR (Polymerase Ch...
See you next time!
Cheers,
Henrik
In the Covid-19 Test:
The primers are designed so that they can just bind to RNA with the exact same sequence of the virus. Then DNA is amplified. Otherwise if the primers don´t match with the RNA, no amplification takes place...
Lot of people does't know how covid 19 testing works share this to others.
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be safe..
And who set the primers? This is just basically guesswork. If you had different primers you had a "different virus". PCR tests are bullshit. There's no way to false-proof the solution of a SPECIFIC VIRUS GENOME.
And before you reply: no it isn't.
nowonmetube your first question can be answered by a failing high school student. GTFO homie
What stops the primers annealing to non-target RNA in a contaminated sample? The number of nucleotides in the primers are extremely small (just 20-30 bases long) compared with the lengths of non-target RNA lengths. The specificity in the presence of contaminants collapses..
@@nowonmetube u have only require one type of primer in case of COVID-19 already its genome ( RNA) is sequenced
So on the basis of this sequenced RNA there are also commercial available primers already designed
No for COVID-19 test
U should have sample from COVID-19 patient
Then extract RNA from it by different isolation techniques now after identification of RNA(Gel etc)
Now we have RNA solution. Now it's time for amplification of RNA by RT PCR
So primer act as marker
I don't know how to thank you... this is what I've been looking for, god bless you! danke schon!
And microbiology homework is done with no stress
SAR-CoV-2 is the virus which cause covid 19. This is diagnosed by rRT-PCR test.
Watch this to know more about covid 19 testing.✅
czcams.com/video/yo9Jaijkjgo/video.html
@@isurusrimadusanka8025 - I just watched that video. It's pretty edited and the narrator isn't a real person (text-to-speach). I don't feel that it is very trustworthy.
Explaining the concept in simple terms is challenging. And you did it great sir🎉
Thank you
This was very well done, thanks for the clear explanation. I think it would be nice to explain more (I'm going to look through your library to see if you have already in another video) how the identification stage is performed. I think I understand but don't feel like I could use this video to help people in my life with the questions I am getting. Thanks again!
Just perfect
Your mRNA sequence is wrong. mRNA is synthesized from 5' to 3'-End (5'--------------3') the mRNA sequence shown at 1:41 is reversed which is wrong.
absolutely amazing video, so easy to understand!
Thank you
I thought the creator of this test said it was not suitable to test for infections diseases???
Because it isn't.
You can set virtually any primers and have multiple different versions of "one virus" with the exact same RNA strange. It's bs.
The SARS-CoV-19 RNA is a genome of this virus and it is also mRNA, on wtich six virus protrins are synthesized
SAR-CoV-2 is the virus which cause covid 19. This is diagnosed by rRT-PCR test.
Watch this to know more about covid 19 testing.✅
czcams.com/video/yo9Jaijkjgo/video.html
I wish you'd take the time to explain the HOW more than the WHAT.
Thank you Sir
Very easily understood your videos
thx for the clear explanation i appreciate this
Fascinating stuff..
Lot of people does't know how covid 19 testing works share this to others.
czcams.com/video/yo9Jaijkjgo/video.html be safe..
The company I work at is in the vital sector. When testing availability increases over the coming weeks, we will have to decide if we will test staff for SARS-CoV2 and antibodies. Thank you for this refresher.
Willem Karet Thanks for sharing this!
This video has bit more about rRT PCR
👉czcams.com/video/yo9Jaijkjgo/video.html
Thank you 💖💖
excelent, thank you fot the video
Is a COVID-19 RNA test a RT-PCR test?
What stops the primers annealing to non-target RNA in a contaminated sample? The number of nucleotides in the primers are extremely small (just 20-30 bases long) compared with the lengths of non-target RNA lengths. The probability that primers will anneal to their composite RNA strands in genetic material, deactivated viral RNA fragments, (INFLUENZA A & B, broken/de-activated SARS-cov-2 RNA strands from previous infection) is very high.
The specificity of SARS-CoV-2 primer protocols in the presence of contaminants within the unpurified snot sample is very small.
This is Real time PCR my friend, completely different from Reverse Transcriptase PCR
Hi, what software did you use for the illustrations?
powerpoint
Hello and thanks Henrik , I appreciate the effort of making your video a lot ! Very easy to understand.
One question. Let's say I got sick with a RNA Virus but I cured and I give my DNA to be tested through PCR for the virus I got cured : Could this technique detect the DNA/RNA of the virus after I am "cured " ?
I want to know if the RNA/DNA remains in me and changes my DNA for ever (without necessarily causing any disease) or what have U read in that regard ?
Immunity is tricky to study genetically, so I'd wager that you cannot use rt-pcr to trace immunity in an efficient, cost effective way. For pathogen immunity, antibody detection is still superior. As for remaining fragments: sometimes viruses do leave trace genome or genome changes in their wake. Thwt's why e.g HPV increases cancer risks in some cases.
@@09csr thank you ! I understand that IgM and IgG through real ELISA tests are the best way to know immunity. I asked it Because in my work there are shrimps that are positive to viruses such as WSSV (DNA virus ) and IHHNV (RNA virus) but never had any symptom of the disease. So my main doubt was that yes some individual can be asymptomatic but the PCR track the DNA of the virus EVEN when if it hasn't expressed 🤨 ?! Thanks ! And hey if U know any book that speaks about that please let me know ,I can only find basic information in my native language 💔
Why do we make cDNA since the 1st step of PCR is denaturation which make ss DNA?
Correct me if I'm wrong, but I believe it's because the virus is an RNA virus. When we separate the cDNA from the RNA strand, it is essentially the same idea as denaturing a double-stranded DNA molecule. The virus does not have double-stranded DNA, so you would first need to create the cDNA from the RNA, otherwise amplification would not be possible.
@@InderSingh-kr2bl Correct
cDNA is more stable than rna .
Excellent Sir.
This video has bit more about rRT PCR
👉czcams.com/video/yo9Jaijkjgo/video.html
Why are the individual bases of the cDNA not complementary to the mRNA but rather just a DNA sequence? Like, the cDNA has only difference in bases such as U with T and T with A's. Other than that, on the cDNA, it's a C for a C on the mRNA and G for a G on the cDNA? I thought it would be complementary and a C on the mRNA becomes a G on the cDNA for example? Apparently from what i checked this is not the case? Can you explain please
Very nice video - stealing for teaching my class!
Knowledge is free, no stealing required! Just be nice and give credit where credit is due.
Very good ❤️
Can someone help me. I'm not that good with science but I roughly understand. During the denaturation of the cda, you use taq polymerase to elongate the the rna strand or the complimentary strand which binds to the rna strand? Also, why can't we detect covid by just amplifying the cdna?
A bit late but the Taq polymerase is used to elongate the single stranded cDNA starting from a hybridized primer. The RNA strand is at this point no longer relevant because it has already been converted to cDNA. Covid19 can't be detected by "just amplifying the cDNA", because Covid19 doesn't generate cDNA. It generates RNA, which we first have to turn into cDNA, like shown in the video.
That will amplify the DNA and we will have DNA in the tube. But then how to decide weather it is corona virus or something else DNA that is amplified.
Pinal chaudhari because the primers just anneal to the sequence on the Corona RNA.. they are designed so that there is no match for other bacterial dna or human dna or whatever.. the primers are very specific
Ok got it
How? They just claim it is lmao
@@henrikslab you know that RNA and DNA is in change all the time, right? Lmao
Great video! One point of clarification here is that SARS-CoV-2 is the name of the virus, while COVID-19 is the name of the disease. So it is really a test for SARS-CoV-2 because you could test positive but be asymptomatic.
are the primers for RNA and DNA the same ?
Why do you make multiple copies of the same segment? Don't you want to just make one or two and move on to the next segment so you get it all reverse transcripted?
The RNA template strand turns to DNA just like that? or de enzyme changes de RNA simple strand to DNA once the enzyme is synthesising? the primer is RNA right? so what changes that RNA into DNA? the same enzyme? you dont explain all of this
How they then identify that the result DNA match the result of what would be obtained from transcripting covid RNA?
Thanks!!😷💞
#Stayathome
Notice how Henricks world did not address Scott's statement
The amount of biology i know is ......when chicken goes on a fryer it gets fried.🍖🍗🍗🍗🍗🍗
Best analogy 👍
Buddy RT-PCR stand for Real-Time PCR. What you are referring to is rRT-PCR
Yusuf Khyalzai Thats not true, but I dont blame you, they get mixed up quite often! www.thermofisher.com/de/de/home/references/ambion-tech-support/rtpcr-analysis/general-articles/rt--pcr-the-basics.html and many other sources say so 😁 Realtime in most cases is shortened with qPCR
@@henrikslab yes you are right.. Q represent quantity of product in real time
This video has bit more about rRT PCR
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Where does the primer come from ? I n the first step annealing
Help
Good one...
Isnt the reagent and primer for RT PCR very commonplace? Then why are we struggling to do many covid19 tests? In Singapore we have thousands of lab techs, hundreds of biology labs and thousands of RT pCR kits each containing enough reagent for 100 samples, but we can only do 3000 tests a day. Why?
What stops the primers annealing to non-target RNA in a contaminated sample? The number of nucleotides in the primers are extremely small (just 20-30 bases long) compared with the lengths of non-target RNA lengths. The probability that primers will anneal to their composite RNA strands in genetic material, deactivated viral RNA fragments, (INFLUENZA A & B, broken/de-activated SARS-cov-2 RNA strands from previous infection) is very high.
The specificity of SARS-CoV-2 primer protocols in the presence of contaminants within the unpurified snot sample is very small.
Does PCR detect the Virus Reading frame region in PCR ? And how ?
Is it as the video said or is it something else.
Thank you for your time
razan abubshait one target is actually in the open reading frame (RNA dependent RNA Polymerase)
Check out the Paper here: www.eurosurveillance.org/content/10.2807/1560-7917.ES.2020.25.3.2000045 (Fig. 1)
The test kits from different manufacturers recognize different regions of the virus genome. Because virus genomes are quite small, there may be up to 6 reading frames to generate all proteins necessary for the virus to auto-amplify in cells. Wether the PCR includes a reading frame or not is not important. The key point is that the PCR only amplifies a portion of the genome that is specific to C19, not portions that could be similar to other coronavirus.
Bobo LRO thank you so much I appreciate it
HenriksWorld thank you so much you’ve been a great help
Is this test for covid19 only or any Coronavirus? Appreciate your answer.
Covid-19 only, since you use primers on sequences that are specific for the virus you want to detect
Right Marco, but with different primers it can also be applied for different other RNA viruses
@@henrikslab Yes of course, I meant that with the SARS-CoV-2 primers you cannot detect other viruses if the primers were constructed properly :)
Sir valuable feedback thanks 20/04/2020
This video has bit more about rRT PCR
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Which primer is used to synthesize the cDNA?
There are many strategies..
There are unbiased approaches:
-often they use random hexamers (this is a mixture of very short primers that cover all possible sequences)
-some use oligo d(T)s, a primer that has only Thymidines to cover the 3´poly-A-tail region
Also, there are more specific ways:
-if you aim for a specific gene instead of an unbiased approach: you can design the primer specific to your gene of interest
@@henrikslab Which tool do you use to design gene specific primers for coronaviruses using the accession number of a whole genome from NCBI? Thanks.
NCBI is great in most cases when designing a primer!
www.ncbi.nlm.nih.gov/tools/primer-blast/
And this exactly shows what the PCR test is completely unreliable.
It only provides a specific set of DNA genomes, not the complete RNA of the whole strand.
thats the point to amplify only genes that are specific to SARS CoV 2
What is primer made of?
They are made from oligonucleotides (or oligomers) which can be synthetically produced or naturally occurring. These are the made from the same building blocks as your genetic material, but are complementary to the genetic material you want to amplify.
To visualize the PCR products, the primers are tagged with fluorescent labels (fluorescent chemicals that bind to the primers).
Primers are small pieces of DNA that match specific portions of the virus genome that are specific for this virus (they don’t match the flu or other virus genome). This is why before creating test kits for PCR it was necessary to isolate the virus and sequence it’s entire genome.
@@BarnsM Thanks for answering!
What is the difference between two step and one step RT-PCR , and what is this?
And what are dNTPs needed for synthesis
@James Candfield --
RE: "and what is this?" --- I assume you are asking what is RT-PCR? Reverse Transcription Polymerase Chain Reaction. It's a method to amplify a sample of RNA by creating a complementary DNA strand.
RE: "And what are dNTPs needed for synthesis" -- these are the nucleotides that you use as the basic building blocks for replication/elongation of genetic material. These building blocks are the four bases: A/T/G/C (or adenosine/thymine/guanine/cytosine) for DNA. Or in the case of RNA, A/G/C/U where U = Uracil.
As the names suggest, the number of steps differ from both methods. In two-step, the formation of cDNA is done in a separate tube. Then only the cDNA is moved to a new tube, and DNA polymerase (plus all other ingredients like cDNA primers, dNTPs, buffers, etc) are added to amplify the cDNA -- this is your typical polymerase chain reaction. But in your one-step, everything is done in the same tube. You put a mixture of Reverse Transcriptase (to form cDNA) and DNA polymerase (to amplify cDNA), along with the RNA primers (to form cDNA) and cDNA primers (to amplify cDNA) as well as the necessary buffers and dNTPs all in one reaction tube. When you throw this mixture in a thermocycler, cDNA gets formed from the RNA sample, then amplified to produce more cDNA. But as you can imagine, the one-step is a soup of different reactions, which can be hard to control for.
Depending on what you need, both procedures have their advantages and disadvantages. Hope this helps!
@@BarnsM Thanks man! Doing me a favor!
How long does the testing process take?
RT-PCR from start to finish (with the sample already extracted from the swab taken from patients) is around 4.5 hours. Up to 1.5hrs to make up the solutions and set up the sample plate (a little longer if you do ddPCR instead of qPCR because you have to create droplets), and about 3 hours sitting on the thermal cycler before the data is ready to be analysed (or 'read' in the case of ddPCR where the droplets have to be fed through a machine to detect fluorescence).
Extraction itself can vary quite a lot depending on what you're extracting and what media it's in, it can be very quick
And what about single cell RTPCR?
This video has bit more about rRT PCR
👉czcams.com/video/yo9Jaijkjgo/video.html
Can we do the test for covid-19 with the classic pcr:conventielpcr. Thanks
Hacene Zermane So they test according to that protocol here: www.eurosurveillance.org/content/10.2807/1560-7917.ES.2020.25.3.2000045
This is basically a classic Reverse Transcription PCR (one step) and then followed by realtime PCR (quantitative PCR) to track the quantity of amplification with fluorescence! If you are interested, check out the published protocol
Yes, the process is a regular PCR. But, PCR works only for DNA, not RNA. Because the genome of C19 is made up of RNA, not DNA, the first steps (RT) simply serve to create a DNA copy of the RNA genome. Once the DNA copy is generated, it is amplified by regular PCR
SAR-CoV-2 is the virus which cause covid 19. This is diagnosed by rRT-PCR test.
Watch this to know more about covid 19 testing.✅
czcams.com/video/yo9Jaijkjgo/video.html
You didn't make clear as to why RT-PCR is useful when compared to normal PCR. You should've clarified that measuring mRNA will yield different conclusions than just DNA.
RT-PCR is used to make cDNA or complementary DNA strand clones from mRNA and these cDNA clones are used to make cDNA library. This technique helps to make selective cDNA from a particular mRNA and amplifies the cDNA clones. In normal PCR we get the multiple copies of particular DNA. After one complete cycle we get 2 copies of the DNA, each of them contain one parent strand and one daughter strand. But in RT -PCR we only get the cDNA clones. This is what I get from my reference.
@@ashnamohan79 Your comment fails to describe why one would opt to measuring mRNA rather than DNA.
Why some time this test give positive and negative to within two or three days
Depend on virus count, there a certain limits where the test can give positive result. You can watch the full explanation on Asian Boss's interview with a South Korean virologist.
Because DNA changes all the time. It isn't set in stone, as people falsely thought it were.
New RT-PCR machine are very nice use laser.... Real time/PCR...
You haven't told much about how to detect Covid through RT PCR?
It's just an imaginative set of primers. If you had different primers, with the same RNA, you'd have a "different virus".
@@KK-lg8uz that's where you're mistaking. Technically speaking yes, if you look at one RNA bit by bit, it's different. But since viruses can EASILY mutate, it can. Also PCR is set only to specific primers. So guess when you have all kinds of viruses with the same primers what happens? Also: Our genes are full of virus RNA! Half of it are virus genes!
@@KK-lg8uz I'm not sure if you understand the process. You have the exact same virus, if you select randomly different primers, you can invent all kinds of new viruses.
@@KK-lg8uz Also, no they don't know what they're doing. There's all kinds of different tests for HIV, depending in which country you are. The test also has changed over the years, several times:
czcams.com/video/uCV8RA5Dx3E/video.html
@@KK-lg8uz you're welcome 😊🐝
Why we use PCR tecnique..?
PCR means amplification of DNA strands so how we know if the virus is present or not..?
And i studied RIA (redioimmuno assey) for antigen antibody reaction so we should useing it for testing the presence of virus..
The PCR is used so we can either
Verify presence (and abundance) of the viral RNA by using Quantitative PCR (qPCR)
or actually sequence the genetic material
I can't speak to RIA that much, they seem like faster and easier to perform but less acurate and take time to create. Theoretically in time we should be able to use them, it is just you have to determine that it works well because the potential for false negatives/positives is higher if you go off of binding rather than just sequence.
Is the narrator German? I love the accent
This video has bit more about rRT PCR
👉czcams.com/video/yo9Jaijkjgo/video.html
Thanks for this. What's the shelf-life like on the materials listed at czcams.com/video/j3ajN0DKaEw/video.html? Is it practical to stockpile them?
Thanks for your question! Shelf-lifes:
RNA sample (frozen at -80°C) ~1 year, however worry about RNases contaminations which are everywhere..
Primers (frozen -20°C) up to several years..
Enzymes (Taq-Polymerase and Reverse Transcriptase) frozen at -20°C up to a year (at least months)
dNTPs (-20°C up to 2 years or so)
SAR-CoV-2 is the virus which cause covid 19. This is diagnosed by rRT-PCR test.
Watch this to know more about covid 19 testing.✅
czcams.com/video/yo9Jaijkjgo/video.html
@@henrikslab Thanks. Not sure why I missed your reply until now.
You video got off on the wrong step right away when he said RT-PCR is Reverse Transcription! So what does the PCR stand for? PCR refers to Polmerase Chain Reaction and this refers to the lab technique to replicate billions of copies of a DNA sample. Trying to explain a complex concept like RT-PCR is absurd on its face and I think your sketchy video is evidence of this.
Michael Deierhoi I recommend you to both listen and watch again before you make wrong quotes or assumptions! I said RT PCR is Reverse Transcription PCR.. be accurate, mate! I further did not explain PCR more in detail since it is clear for most biologists already and as I also mentioned in the Video.. I already did make a video on PCR. Cheers
@@henrikslab Okay I stand corrected. You did in fact mention what PCR stands for at the end of video. Better late then never as the saying goes.
Yes this video needs to be amended heavily or removed! Very inaccurate
Misleading Video. No elaboration, no explanation.
This video has bit more about rRT PCR
👉czcams.com/video/yo9Jaijkjgo/video.html
Propaganda
Thank you