All about real-time PCR (qRT-PCR). How it works? Made Easy.
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- Опубликовано: 25 июл 2024
- Most exhaustive information about RT-PCR, types, working principle, requirements, advantages, applications will be covered in this video with a special emphasis on Covid-19 detection.
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Language is the reflection of our real character. Kindly, think before posting. Our views may differ and will differ. At times I may be wrong and you may be right but at times, it may be the other way round. This does not mean we should disrespect each other. Using unpleasant words will not prove anyone right.
Disrespect is a weapon to attack people who present arguments they can't prove wrong. I love your humility!
Hey I'm from Taiwan. Thank you for this detailed information! I really like the comparison tables in the video.
@Sophie. Thanks
Amazing video ..Grateful 🙏
Nice information.
excellent video
Great video easy to understand
Thanks
it is a good video. You have been picked upon by the crazies. I would disable comments and let them comment on their own videos
Nice video
Thank you
Ur all lactures was amazing whom i visited thanks dear sir
Thanks so much.
Thanks, I learnt something, although I already knew the basics of the RT-PCR test. I did not realize there are different methods being used, So many saying it's not accurate which goes against our experience in NZ. We have done a total of
Glad it was helpful!
@Storm November Show your logic. I will say this, there is not just one PCR test, there are many different primers etc that are used, each needs a different threshold. sometimes here, the say there was a "weak positive", a test with a high Ct number. It is either someone who has caught it in the last few days or a "historic case". the do another test later to find out. I can't speak for every other country but here it's spot on!
@@willgeorge5644 in mnn. Jjhgfghhhhhhhh hmm hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhghhhggggggghhhhhhhhhhhhh gg gghnnnbbbbbbbbbbb
Understood the concept
Love your lecture
thank you'
बहुत अच्छे से समझाया
Thank you
Thanks
Question: How can you have a test with such a large variance (in fact ANY variance) in the cycles for amplification? In order to have consistency and repeatability, you cannot have anything that is arbitrary. How were the number of cycles determined?
Variance mainly depends upon your sample and handling. You can minimize it. what do you mean by arbitrary here? You use specific primers and probe to amplify and detect the target. PCR cycle at which you record the signal as significant or Ct cycle is your cycle number for that sample.
Hope this would help.
@@XploreBio The graph show Ct from 35-40 cycles... why the range? I said arbitrary because it is not standardized or there is no reference as to how ___Ct was determined to be the proper Ct. Thanks for responding.
Ct values will change with the nucleic acid template you take. But if the recommended template conc. is taken, and still Ct value is beyond 35, it suggests the target gene is expressed very poorly or not at all. Same is the case with Covid 19.
@@XploreBio How was 35 Ct determined to be the cutoff point? Is this test actually observing the Covid-19 virus or is it observing associated enzymes with Covid-19? Thanks again.
Normally with any RT PCR reaction with any gene or sample, Ct value must be below 35. If its above 35 it means low conc. of target mRNA in the sample. It is identifying Covid 19 RNA using primers and probes targetting gene for spike protein.
Thanks for your effort.
I have a question pls
Why do we need billions of copies to check if we have the virus ?
Why not comparing only one sequence of the extracted RNA or check only with one cycle if the primers will bind ?
You need to have sufficient number of RNA or DNA copies to be detected by the instrument. Real time PCRs can not detect a single RNA/DNA molecule. You need to amplify the template so that it can produce sufficient fluorescence to be detected by the instrument. I am sure in future we can have instruments that may detect single molecule of nucleic acids. Thanks for asking.
@@XploreBio
Thanks for your reply.
I saw in several tutorials that fluorescent light is emitted in every cycle. Doesn't this mean that we can detect RNA from the first replication ?
No, because the signal is too low to be detected at first. Below detection limit.
@@braveheart2205 Basically the test is getting a piece of extremely small material to fluoresce (light up) if it's the type of material we're looking for. If there's not much of it, then it won't light up bright enough to detect. If you keep doubling it and checking for fluorescence each time, you can determine how much of it there must have been at the beginning because you know how much of it you need before you can see it.
Detail: I'm not sure whether it actually "doubles" (multiplies by 2) each step, but the way exponential functions work, it doesn't really matter what the multiplying factor is, you can think of it as doubling some number of times. For example, if each step amplifies by 1.3, and some sample took 20 steps to detect fluorescence, that's the same as taking about 7.5 doubling steps. In any case, that's not super important to the explanation. =)
Thank you so much brother for such nice and simple explanation. One thing you have used gene expression I am still confuse why you use it while explaining PCR? Please answer
RT PCR estimates transcript level of gene. Hence, I used term gene expression. Although, actual gene expression is represented by proteins. Hope that answers your query.
@@XploreBio thank you for reply, in a sense of making DNA from DNA ( invitro replication) you used gene expression am I right dear ?
@Qaisar's Biology. We can not directly quantify individual mRNA for two reasons: 1. concentration of individual mRNA (gene) expressed is low to be detected. 2. We need to specifically target that mRNA or its cDNA using its specific primer for this PCR using set of specific primers and probe is done.
Here invitro replication is not done to study expression rather to target and amplify sigbal of target mRNA or its complementary DNA.
@@XploreBio thank you brother
The test was invented in the 1980s by Kary Mullis who won the Nobel prize for chemistry as a result.
However, he said it was for research purposes only and should never be used for diagnosis.
Mullis died last year but there is still video of him talking about the test and saying
"If you do it well you can find almost anything in anybody".
If the government had not decided it must "test, test, test" it would not have found any justification for
restricting freedom.
True.
There is a retraction for drosten corman paper sent to eurosurveillance cormandrostenreview.com
Edit: This is the scientific base of the whole fraudulently employed PCR test.
Glad to see some comment from normal people here. Yes Kary Mullis is a big story.
This "test" cannot tell anything about the quantity of pathogen in specimen. How can it be used for diagnosis?
The most ridiculous part is that the DNA primer is not based on the genetic sequence of a true virus, but a computer simulated RNA model.
The current way using this technology looks like a fraud, rather than true science.
What you and many others fail to understand and this example avoids telling you is that KaryMullins the inventor specifically said that the PCR testing is not meant for nor reliable for diagnosing any specific disease. There are actual diagnostics for that. Many are running the cycles so many times, especially beyond 10 until it will give you a positive test result.
sir if possible please do a dedicated video on intercalating dye based rt-PCR
3:18 JON JONES!
Is it Real Time or Reverse Transcription?
Better to say reverse transcriptase PCR if reverse transcription is followed by pcr amplification. But most of the times reverse transcription is separately done and then PCR. I use RT for quantitative Real time PCR. Reverse transcriptase in full not short to avoid confusion. But everyone use it differently.
RT-PCR means Reverse Transcription PCR only na? I think in Covid testing ts qRT- PCR , since ts a combination of qPCR (quantitative real time PCR ) and RT-PCR
The kits for cornavirus qRT PCR do reverse transcrition and PCR too. therefore you can say RT PCR here as reverse transcription PCR.
@@XploreBio good video..nicely presented👌
The rest does not identify viruses.. only genetic sequences or proteins quantified in cycles to make it look like something, when it’s not.
Right. The test quantifies Viral genetic material.
@@XploreBio Interesting quote from the inventor of the PCR test Dr Kari Mullis PhD. “Quantitative PCR is an oxymoron. PCR is intended to identify substances qualitatively, but by its very nature is unsuited for estimating numbers. Although there is a common misimpression that the viral-load tests actually count the number of viruses in the blood, these tests cannot detect free, infectious viruses at all; they can only detect proteins that are believed, in some cases wrongly, to be unique to HIV.
“The tests can detect genetic sequences of viruses, but not viruses themselves.”
@@XploreBio Anything above 17 amplification cycles increases the chances of a false positive.
u missed out the step of conversion of extracted RNA samples to cDNA before qPCR
check at 1.47 min
It does not test for virus, FULLSTOP.
Read: Principles and applications of polymerase chain reaction in medical diagnostic fields: a review.
Real-Time PCR is extremely useful in the study of viruses that cause infectious diseases. The majority of published assays show an increase in the frequency of viral detection. This is therefore an attractive technology for many virological fields (56). It is valued for its quickness in the detection of viral variants and the syndromes caused by these viruses (27). The method contributes to epidemiological studies due to its capacity to quantify nucleic acids in a single reaction (46,120). New chemicals have allowed a better discrimination of multiple viral genotypes within a single reaction (44) and have provided an alternative viral detection method based on morbidity and mortality assays.
Rather for Nucleic acid target.