RT-qPCR for diagnosing COVID-19 (former 2019-nCoV) 

Me too... I am a PhD graduate who joined academia right after graduation, and spent 4 years in teaching and administration :( This video brings back the good memories of my PhD journey...

I really appreciate that, hope you get to do that.

RNA can be amplified?

cls new you have to create cdna molecule from it first

@@beaa8153 yes for that reverse transcriptase is needed

Hi Octav, you make a really good point and you certainly can 'pool' lots of samples together to speed up testing. If your 'pool' then comes back positive, you will have one or more individual samples which has tested positive, which you can then test individually. This type of technique works best when you have a low incidence of a disease in a community, although it is a good way of identifying asymptomatic infections. At the moment, most countries have more cases than they can test, so are more likely to concentrate on diagnosing individuals (e.g. patients in hospital or front line staff).

@@pandora-id-netconsortium1846 thank you for your answer. I am glad to see that this is a real possibility and I would think that trough this method you could diagnose not individuals but entire communities and quarantine them or at least raise the awareness that there is a a high risk and have people take precautions. With test so scarce these days, I would think this method could save lives. Once again thank you for your answer.

Hi Putra, you're correct, this is real time PCR. It is also known as quantitative PCR. The naming gets confusing, as this is reverse transcriptase (because you are turning RNA into DNA) real time PCR. So in actual fact its full name would be, RT-RT PCR, or RT-qPCR. This starts to get a bit long, so it is usually just called RT-PCR, or sometimes RT-qPCR.

@@pandora-id-netconsortium1846 Thank you very much for the reply :)

@Monky Hi. I think ELISA is used to detect HIV

@@jayagopalkrishna8790 confirmatory is RT PCR..or western

@Monky for HIV the Ab detection is more reliable. As the virus may not be found in blood for long time. Usually ELISA or western blot is sufficient

Hi Ain, PCR cannot amplify RNA, so we must make cDNA first, using the viral RNA as our template. In this video, we show one step RT-PCR, where you create the cDNA using reverse transcriptase and then amplify it using polymerase in the same tube. There are also two step kits, where these steps are done separately. So yes, it is the cDNA that is considered positive, but you have to make it before you can amplify it.

@@pandora-id-netconsortium1846 Thank you!

Hi Jorges, as a minimum, you would need to run a negative control (e.g. a water sample processed from the beginning with the real samples) to check for (cross)contamination and a positive control (e.g. confirmed SARS-CoV-2 nucleic acid) to ensure the results that you are seeing are the right thing. Often more controls are run for extra confidence, but in theory the maximum number of samples you could run on a 96 well PCR machine is 94.

@@pandora-id-netconsortium1846 Hi, as each sample should be run in triplicate or at least in duplicate, the number of samples tested is reduced to 30 or 46 per 96-well plate.

Is this a hard test to perform?

GENE IS RT PCR AND PROTEIN RAPID TEST

Yep, dont forget to amplify it correctly, if you dont, good luck finding out if you have a false positive, or a true positive which if you get it high enough, everyone can test positive. Once you find out what the origin is for what you are testing for, you will understand why.

No its the same

@Nenad Ilić what is the role of the probe? Is it tagged with fluorescent matterial?

because they have a lot of pharmaceutical factories they already had the infrastructure in place to do mass testing - not quite sure if this is correct but hope it helps anyway lol

Hi Imee, Cepheid got emergency approval for a SARS-CoV-2 cartridge for the GeneXpert very recently. As this is classed as an 'emergency' roll out, the sensitivity and specificity of the test will likely need to be validated. The GeneXpert does utilise PCR, so it works in a similar way. Cepheid have some good videos on RU-vid about how it works.

Hi Joshua, this is likely to depend on the laboratory processing the samples. There are automated extraction and PCR preparation machines available, but it would depend on the size and income of the laboratory. As this video is aimed providing information to Low and Middle Income countries, such as those in the PANDORA-ID-NET consortium, we wanted to show all of the individual, manual steps.

@@pandora-id-netconsortium1846 thanks for your reply. I was a lab researcher for several years, now a high school biology teacher and was curious about how samples are processed and how throughput has changed. Thanks again.

@@joshnelsonnj Hello Joshua. I've been working in a testing lab for the last year, We still handle some samples manually, but most are lysed in deep (96) well plates using robotic liquid handlers which also add magnetic beads and phage (extraction control). Extraction (via the beads) is automated as is mastermix prep and prep of PCR plates. We do approx 40-60K a day between 150-200 lab techs.

Hi Imee, serology tests are also being developed currently. Serology tells us if a person has been exposed to the SAR-CoV-2 virus, but it cannot tell us whether it is a current infection or not, because it is looking for the person's immune reaction to the virus, which continues after the actual infection and virus has gone. Serology is good for identifying how many in population has contracted the virus during the outbreak, or whether a person (such as a doctor) has already had it (perhaps asymptomatically) and therefore would be able to continue working. In comparison, PCR tests that identify viral nucleic acid tell us whether a person has a current infection. So they have slightly different uses, but are both still very helpful in an emergency situation like this.

@@pandora-id-netconsortium1846 what are the possible ways that the virus can be spread in the laboratory

@@pandora-id-netconsortium1846 I have a challenge for you. Try testing things like say, apples, pineapples, oranges, water from the tap, watermelon, (raw chicken eggs those for sure) or anything else they use in flu vaccines Be sure whatever is used was fed GMO feed laced with Glyphosate/Round up. etc. Use a variety of things. Run those at 40 cycles and see what happens. If you can get your hands on any flu vaccines from last year late 2019 test them as well to see what is in them. Vasculitis is caused by a few vaccines. They found that to be a finding, in many of the Autopsy reports from Covid Patients that died. These vaccines are the one I am most interested in. Afluria, Engerix-B, Fluarix, Fluzone, Havrix, MMR-II, Recombivax, Twinrix

@@kiethz8201 you forgot mayonnaises!

Hi Oğuzhan, The risk to laboratory staff is very low, as long as they follow their local health and safety protocols whilst processing these samples. SARS-CoV-2 has been designated a hazard group 3 pathogen, so the extraction of viral RNA must be done in a BSL3 laboratory within a microbiological safety cabinet to protect workers from aerosols (who must also wearing the appropriate personal protective equipment). Once the RNA is extracted, no live viruses will be present (as they are broken open to extract the nucleic acid), so the resulting samples can then be moved to a BSL2 laboratory for the rest of the protocol (quantifying and PCR). The RNA extraction part of this protocol should only be undertaken by laboratory staff who are trained to work in a BSL3 laboratory.

The sensitivity of the test relies on many factors, for instance the extraction method, which kit you use and what gene your primers target. This is why it is vital to validate your testing procedure using standardised controls etc. PANDORA are creating a Global Health Network hub specifically to provide SARS-CoV-2 diagnostic test validation guidance. You may find that the two laboratories you speak of are doing entirely different protocols and if one is poorly validated compared to the other, they will have vastly different specificities and sensitivities.

Hi Maha, do you mean the whole process? This depends on how many samples your are processing at a time, and also whether the process is automated at any stage. If you are doing it all by hand, as in the video, the RNA extraction is likely to take around 1 hour (plus then quantification of the nucleic acid), then the PCR stage (setting up the reaction and running samples on the machine is likely to take another 4 hours

Exactly. Scamdemic it is.

Hi Anna, these are good questions! The good thing about real time PCR is that it does provide you with a visual representation of the data at the end. Once a procedure has been validated, and as long as you use positive and negative controls, identifying positive and negative results is fairly straight forward (i.e. does the curve for a particular sample have the same shape as the positive control and does is rise above the negative control lines (which should be flat?) Some RT-PCR programmes will automatically provide results if you set it up to. Results can be subjective though, if you have a very small viral load to begin with, it will take much longer for the line to rise above the background level (or 'reach the cycle threshold'). Laboratories typically set a CT value cut off (usually around 35 cycles), so that any sample with a CT value of higher than that is considered a negative (background fluorescence can start to amplify after a lot of cycles). So whilst RT-PCR machines (and good validation of a specific test) mean that it is usually fairly simple to tell positive from negative. It is not always the case. Depending on the test you set up, RT-PCR machines can be semi quantitative or quantitative. Whilst diagnostics and clinical trials are governed by strict quality control, often more research based science (and depending on the tests used) can be very much opinion based. I hope you found this useful, please don't let negative comments like the one below put you off!

@@beamarie8041 There's no need for comments like this

@@linzyelton4592 Yea you're right so I took it down. There have just been so so many unbelievably rude conspiracy comments on this video and countless others that I started getting frustrated and pissed off thinking this was another one. Sincere apologies to OP, I misread the comment and the rudeness was for the conspiracists lol.

Maria Taylor what you wrote? I agree there is many conspiracy theorist on testing. They are small so it is ok. (I am learning english apologies)

Maria Taylor the question is fair but I see how you can mistake it -> getting angry and responding to conspiracy comments is useless - they are annoying, but harmless...

Hi Ibrahim, RT-qPCR is preferred as a diagnostic tool, because the results are more sensitive and specific. However, where RT-qPCR is not widely available, conventional PCR could be used as a pragmatic tool, as long as appropriate validation of the assay has been undertaken. Different countries have different rules as to what diagnostic tests are acceptable, so you should check if classical PCR is approved for diagnostics in Algeria.

Hey dude, im from algeria as well. Is it true that only Pasteur's institution got a qPCR?

To our knowledge, there is so far no evidence that nasal wax or nasal spray would prevent or treat COVID-19. In any case, any treatment should be tested in a controlled environment, i.e. as clinical trials and published in respected peer viewed journals before being delivered or used by the community. Viruses can be spread through mucous membranes including the mouth, so you would need to protect all potential entries into the body.

hay un enlace en la descripción del video al articulo publicado. ella sigue el protocolo

Estamos trabajando en una versión en español, sigue revisando nuestro canal de RU-vid

Gregoire Riviere gracias ya lo vi 👌🏽

PANDORA-ID-NET Consortium gracias por la aportación , pude traducirlo perfectamente 👌🏽

Abhi Bhadran duhhh boy! We are extracting any random RNA from human swab and identifying covid 19 rna through the covid 19 probe

There testing symptoms? Who let this happen!

@@fabriciooliveira3720 so I could litterally have any infections or viruses and I'll have a good chance of testing a false positive?

RT-qPCR is very sensitive, as in theory you only need one strand of DNA for the primers to pick up and amplify. There are a number of different protocols which are used and adapted by different countries, all of which vary slightly, but they will have all been validated to ensure that they are suitably specific and sensitive for SARS-CoV-2. They will have positive controls and negative controls, potentially including controls against human DNA, ensuring that it is only viral RNA that is being picked up by the primers. The primers have been designed to be specific for the sequence in SARS-CoV-2 genes (scientists search for sequences in a massive database to ensure that they are unique), so will only pick up this virus' nucleic acid pattern. This means that it will only pick up SARS-CoV-2 and not any other virus.

@@fabriciooliveira3720 Perhaps it would be useful for you to provide your reference for this comment, so that it can be justified?

@@pandora-id-netconsortium1846 The issue isn't necessarily the sensitivity of your test (and primers in this case, which is debatable), but the quality of our specimens, when I have time I'll post some of articles the here, but the general consensus from a few articles is a 30-71% sensitivity of our current RT-PCR if we collect NP-throat swabs (even early in the disease when VL is relatively high in that site)

Yes it is

Cuz these test kits are not supposed to be used for diagnostic proposes but for research.

RT-qPCR can result positive or negative depending if a cDNA fragment of interest is present or not. If there was no viral RNA, then there would be no template for cDNA, and despite thermocycling, it would result negative. Like the negative control shown in the video.

Nope. It never was/is infectious. The way they proved polio caused paralysis was by continuous applications on many animals with no evidence. It was until they drilled monkeys in the head and Injected a cup and a half of virus filled water. One died, the other was paralyzed, then died. If this is the science people are IGNORING, I can see why...

@@vincevasquez5841 but after polio vaccine this paralysis has been reduced dramatically

Hi Dr Osama, you will find all the accompanying information on our TGHN pages: pandora.tghn.org/covid-19-diagnostic-tools/molecular-diagnostics/ and a link directly to the protocol from the video here: media.tghn.org/medialibrary/2022/02/Outbreak_diagnostics_SARS-CoV-2_-_Protocol.pdf

She said real time RT PCR

What is the difference between real time PCR with reverse transcriptase PCR

🤣 As stated she said 'real time RT PCR' That's to say *real time reverse transcription polymerase chain reaction* Oh dear 🤦🏻‍♂️ You really wouldn't know a reliable video would you 🤷🏻‍♂️

@@khgnew763 It's not really a question of difference. RT- PCR stands for Reverse Transcriptase- PCR. We call it that because we use reverse transcriptase enzyme to make a complimentary DNA from the viral RNA. This is necessary because we cannot amplify an RNA alone in a PCR. Amplified DNA tells us indirectly that the sample is positive for viral RNA that means the patient is positive for the virus, i.e., coronavirus in this case. As far as the word 'Real-time' is considered, we call this Real Time PCR because on the screen you can see the progress of the Polymerase reaction in real time and the detected viral load increases (the line on the graph rises) as time passes if Coronavirus was present in the sample. Hope this settles it.

@@hammadmufti4007 greatly explained

Why so. It's the most accurate one as we target the genes directly

Craig Doe Because it says so in the insert. It is an amplification technique not a diagnostic one. The inventor himself said as much but he’s no longer alive as of a few months ago to refute its use as he did when it was being used for hiv testing and ‘viral load’ counting. He wrote a paper titled ‘Viral Load Of Crap’ as a matter of fact. Dr Kary Mullis. A great scientist and principled human being. Look him up.

@@esaimorales814 I totally agree!! Makes no sense, even using this for the Pandemic. its crazy stats going on because of this. Do you have that Paper he wrote, or you have a link?

Why she didn't wear mask ...

Hi Sundar, whilst SARS-CoV-2 is quite a large virus, viruses in general are too small to see with a conventional light microscope. You can see them using electron microscopy, but unfortunately this isn't practical for large-scale diagnosis (they are expensive and take up huge amounts of space).

noo

Maybe exosome

Shhhhh....dont let the cat out of the bag

Before proceeding, I think it's important we get the terminology correct: COVID-19 is the disease, SARS-CoV-2 is the etiological agent (i.e. the virus). So you would isolate SARS-CoV-2, not COVID-19. Virions of SARS-CoV-2 have been isolated, the RNA extracted and amplified, the subsequently sequenced across many samples. You can check the publicly available NCBI website, where the sequences of thousands of samples from throughout the world have been submitted and can be downloaded (as FASTA files, a type of .txt file). For fun, you can take these files and put them into a phylogeny program (there is free software for this) to form a parsimonious tree to approximate the phylogenetic relationship of existing sequenced samples. Sequencing technology is quite rapid compared to what it was only a 2 or 3 decades ago. For example, in a small project investigating some novel bacteriophages (viruses that infect bacteria) that I was involved in had 2 of our phages sequenced and data retrieved in under 2 weeks. The sizes of those 2 phage genomes were 50 kilobases (kb) and 150 kb long, both of which are significantly larger than that of SARS-CoV-2 (only ~30 kb).

Hákon Sigurðarson THANK YOU...But they won’t understand. They don’t want to understand.

Nenad Ilić and the earth is flat because no human while on earth can see it as round. Astronauts are paid actors, alex jones said so!

How this virus can spread in the laboratory?

Hi Mayank, this was filmed in a UK BSL3 laboratory. The health and safety requirements in the UK mean that there is a strong negative pressure gradient moving away from the user and out through a HEPA filter in the microbiological safety cabinet (MSC). As you can see, all work in the video is being conducted within the MSC and therefore laboratory workers are not exposed to aerosols etc. unless there is a spill outside the cabinet (there are emergency procedures in place for this). So there is no need to wear a face mask, because you are protected by negative air pressure. The BSL rules differ depending on your country and also the level of laboratory (currently, according to the WHO, SARS-CoV-2 samples can be processed in a BSL2 laboratory within an MSC, but any culturing must be at BSL3 level). It may well be necessary to wear face PPE in many laboratories if the health and safety standards are different (we work with many countries setting up BSL3 laboratories and the standards vary hugely). This is is something you must check before undertaking any pathogen work.

@@pandora-id-netconsortium1846 if we use bsl2 safety cabinet for extraction we don't need face masks?

Mask is not needed. She's using a biosafety cabinet(negative air pressure + HEPA) google to see how they work. Airflow is pull away from her

@Nenad Ilić You're correct, there are two types of cabinet, a class I (which primarily protects the user) and a class II (which protects both the user and the sample. As Eri said, in the UK, for BSL3 laboratories, we use negative pressure systems, which drag airflow away from the user and out through the top of the microbiological safety cabinet through a HEPA filter. So the user is never exposed to aerosols if work is done within the cabinet. Because of this, in the UK, masks are not required. However, these health and safety rules can vary a lot between countries and some do require masks (because they rely less on negative pressure, which can be expensive to set up). Ebola is a BSL4 pathogen and requires even greater health and safety requirements. The current WHO guidelines state that SARS-CoV-2 samples can be processed in a BSL2 laboratory within a cabinet, but culturing of the virus must be at BSL3. Hope that helps

Artem Rodichkin wait, what?😳🤦🏼‍♂️🤦🏼‍♂️🤦🏼‍♂️🤦🏼‍♂️

There are cheaper available methods for comiting homicide.