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The story of Kary Mullis who won the Nobel Prize in 1993 for creating the reverse transcription-polymerase chain reaction (RT-PCR) test which is today used for COVID-19 tests.

Quite a gripping read although I zoned out in the bits about polymerised RNA dosbodisation. 

He always felt that 30 amplification cycles was the top limit and was opposed to 40 cycles which is in use for COVID-19 testing. This awkward objection was solved however when he sadly died in 2019 just before global virus deployment, er I mean accidental release, er I mean unfortunate bat soup incident.

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1 hour ago, Funn3r said:

He always felt that 30 amplification cycles was the top limit and was opposed to 40 cycles which is in use for COVID-19 testing.

Depends a bit on the design of your oligonucleotide primers and probe and the sequence of what you are trying to amplify, with the best will in the world it's not always possible to get a low copy sequence to amplify up in under 30 cycles, some sequences are just a bugger to PCR. It's true that lower cycle number is generally better though, less chance that some second off-target sequence which your primers happen to bind to weakly will amplify and give a false positive signal. You can control for this by doing melting curves though as if you've amplified a false positive it's extremely unlikely to have the same melting profile as the thing you were actually trying to amplify. 

Here's an example of cycle times seen with a qPCR for real clinical Covid-19 samples, looks like they're mostly but not all coming up below 30 cycles (N1 and N2 is Covid-19 target sequence):

https://www.biorxiv.org/content/10.1101/2020.04.02.022186v1.full.pdf

image.png.6c2e4ea641bac47b0310f8e5bfa61102.png

Edited by Darude
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1 minute ago, Darude said:

Depends a bit on the design of your oligonucleotide primers and probe and the sequence of what you are trying to amplify, with the best will in the world it's not always possible to get a low copy sequence to amplify up in under 30 cycles, some sequences are just a bugger to PCR. It's true that lower cycle number is generally better though, less chance that some second off-target sequence which your primers happen to bind to weakly will amplify and give a false positive signal. You can control for this by doing melting curves though as if you've amplified a false positive it's extremely unlikely to have the same melting profile as the thing you were actually trying to amplify. 

Whilst that is true of true low copy targets (minimal residue disease leukaemia DNA translocations for instance, or low viral load), for something like covid-19 which I would presume to be reasonably frequent target sequences then 25 cycles is more than enough.  You should be seeing exponential rise in PCR product by 5-10 cycles and then in the exponential growth phase up to 20 cycles.  After that I would be thinking your PCR reactions aren't optimised in terms of primer/reaction/annealing temperature conditions (usually magnesium concentration).

PCR is an error prone process and should only be used as an indication, the gold standard of course being confirmation by sequencing - too expensive for large scale population testing.  Antibody reaction test would be preferable in a clinical diagnostic setting

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7 minutes ago, JFK said:

for something like covid-19 which I would presume to be reasonably frequent target sequences then 25 cycles is more than enough

That's a pretty big assumption, lots of reasons why the Covid-19 target frequency would vary a lot in real world samples e.g. stage of disease and severity of infection, volume and type of patient sample from which the RNA was extracted etc.

Edited by Darude
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3 minutes ago, JFK said:

PCR is an error prone process and should only be used as an indication, the gold standard of course being confirmation by sequencing - too expensive for large scale population testing.  Antibody reaction test would be preferable in a clinical diagnostic setting

There are alternatives to PCR other than sequencing e.g. LAMP (which in fairness is pretty similar to PCR):

https://www.technologynetworks.com/diagnostics/blog/lamp-based-testing-for-covid-19-340508

or CRISPR-Cas12a-based methods (too experimental at the moment really):

https://www.nature.com/articles/s41587-020-0513-4

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7 minutes ago, Darude said:

That's a pretty big assumption, lots of reasons why the Covid-19 target frequency would vary a lot in real world samples e.g. stage of disease and severity of infection, volume and type of patient sample from which the RNA was extracted etc.

It is, but I wouldn't compromise my assay for the sake of the variability in target sequence, either target sequence enrichment (specimen source) or more specificity, I'm not too sure on the specificity of the PCR, there's always going to be that nagging doubt of non-specific primer binding - hence confirmatory testing (sequencing etc)

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5 minutes ago, Darude said:

There are alternatives to PCR other than sequencing e.g. LAMP (which in fairness is pretty similar to PCR):

https://www.technologynetworks.com/diagnostics/blog/lamp-based-testing-for-covid-19-340508

or CRISPR-Cas12a-based methods (too experimental at the moment really):

https://www.nature.com/articles/s41587-020-0513-4

Interesting, I remember trials of iso-thermal amplification methods, now this was on human genomic DNA I was in the field of so the non-specific binding meant these assays weren't robust enough for our work (molecular genetic diagnostic testing). 

Could be useful for pathogen detection, obviously would need to be specific - there's an issue with coronavirus I suppose, trying to find that strain-specific region, then issues of viral mutation ... I'm not sure it's going to be a starter, in terms of cost and issues with viral mutation.  Probably a robust, standard test like an ELISA type detection method for antibody reaction would be more feasible. 

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4 minutes ago, JFK said:

It is, but I wouldn't compromise my assay for the sake of the variability in target sequence, either target sequence enrichment (specimen source) or more specificity, I'm not too sure on the specificity of the PCR, there's always going to be that nagging doubt of non-specific primer binding - hence confirmatory testing (sequencing etc)

Could always confirm by nested PCR if melting curves aren't enough and sequencing is unavailable/impractical.

Edited by Darude
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26 minutes ago, Darude said:

Could always confirm by nested PCR if melting curves aren't enough and sequencing is unavailable/impractical.

True, but I would question the validity of the whole testing procedure - this would be fine for something specific and low copy number (genomic translocations in minimal residue disease for example) where the target is unchanging, but probably not realistic for a diagnostic test for population levels

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Can you explain it to a layperson... Is it right to say this test in the context of the alleged pandemic is a flawed approach and deliberately oversensitive to boost the statistics and increasing number of "cases"?  Or not?

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10 minutes ago, Funn3r said:

Can you explain it to a layperson... Is it right to say this test in the context of the alleged pandemic is a flawed approach and deliberately oversensitive to boost the statistics and increasing number of "cases"?  Or not?

In my opinion it's not correct. I've done plenty of dilution curves where you take a target you are trying to quantify and make weaker and weaker dilutions of it, then stick them on a qPCR machine. The more concentrated samples come up below 30 cycles and the most dilute samples come up exactly where you would expect them to be between 30 and 40 cycles and the negatives are negative. Saying that beyond 30 cycles you are no longer quantifying real target is a pretty extreme position and is underestimating the sensitivity of a good qPCR method.

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11 minutes ago, Darude said:

Saying that beyond 30 cycles you are no longer quantifying real target is a pretty extreme position and is underestimating the sensitivity of a good qPCR method.

So the dodgy-looking "NHS Guidance" recommending 45+ cycles is OK and actually not dodgy?


image.png.82179d754f808d003972382383fe9c7c.png

 

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5 minutes ago, Funn3r said:

So the dodgy-looking "NHS Guidance" recommending 45+ cycles is OK and actually not dodgy?


image.png.82179d754f808d003972382383fe9c7c.png

 

The NHS guidance document you linked (page 9) tells the technician that in the event of amplification being detected at 40 cycles or more that this shouldn't be reported as a positive result and the test should be repeated. Seems a reasonable position to take.

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8 hours ago, MrPin said:

When you say PCR, I imagine the old "mess room" radio. Listening to the Forces Service whilst stuck in the middle of Bongo Land

http://www.vintageradio.me.uk/military/prc2_a.htm

Funny they make these appliances so huge with lots of empty space inside

I have a VHS machine like that

 

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