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This is good cast from @StephenABustin.
@Eurosurveillanc should listen to this.
I agree with Stephen on most topics in this but I take exception to his claims that our retraction request is an "utter disgrace" while also admitting to not looking at these primers. Lets dig
@Eurosurveillanc should listen to this.
I agree with Stephen on most topics in this but I take exception to his claims that our retraction request is an "utter disgrace" while also admitting to not looking at these primers. Lets dig
Here is the cast.
http://chironreturn.org/audio/210201-steve-bustin.mp3
Dr. Bustin is the lead author on a very important paper related to standards in PCR known as MIQE.
It's refreshing to see part of the interview where Steve is open to read new papers and change his mind.
http://chironreturn.org/audio/210201-steve-bustin.mp3
Dr. Bustin is the lead author on a very important paper related to standards in PCR known as MIQE.
It's refreshing to see part of the interview where Steve is open to read new papers and change his mind.
I’m hoping he will consider reading our Addendum to the CD protocol as I think he will also change his mind once some of the details he was missing is presented.
I think he will find the Ethos of MIQE threaded throughout this dissection of the CD PCR. https://zenodo.org/record/4433503#.YBiDrC2z1Yj
I think he will find the Ethos of MIQE threaded throughout this dissection of the CD PCR. https://zenodo.org/record/4433503#.YBiDrC2z1Yj
Before we start this.. It is important NOT to conflate the CD PCR protocol with other PCR protocols Dr. Bustin may be familiar with as most don't fall into these traps and he will probably be shocked to learn the CD protocol does not meet MIQE or his own publications on this.
Spoiler Alert. Stephen mentions he was one of the @Eurosurveillanc reviewers to our retraction request. Why is his work not public? He shouldn't have to mince words about this in a cast. Secondly, you will find parts of this cast where he admits to not having looked closely at it
It's quite possible, he only reviewed the initial retraction request and didn't see the addendum and that is why the reviews are not public?
Lets go minute by minute as there is plenty we agree on.
Lets go minute by minute as there is plenty we agree on.
8 mins in, Stephen brings up PCR inhibition and I'm glad he covered it.
Some twitter paraphrasing.
"If you have the proper controls you can address inhibition".
CD doesn’t have internal controls.
Heme is a good point. It chelates Mg2+. Does any end up on the swab?
Some twitter paraphrasing.
"If you have the proper controls you can address inhibition".
CD doesn’t have internal controls.
Heme is a good point. It chelates Mg2+. Does any end up on the swab?
10:00 in.
Calibrate the test to infectiousness.
Stephen claims "this hasn’t been done yet”.
Yes it has…but we agree each test needs to recalibrate Cts to this... Jaafar et al.
We agree 2 tests at different time points would answer this as well. https://academic.oup.com/cid/advance-article/doi/10.1093/cid/ciaa1491/5912603
Calibrate the test to infectiousness.
Stephen claims "this hasn’t been done yet”.
Yes it has…but we agree each test needs to recalibrate Cts to this... Jaafar et al.
We agree 2 tests at different time points would answer this as well. https://academic.oup.com/cid/advance-article/doi/10.1093/cid/ciaa1491/5912603
11:00 min in,.
A single Ct is Not viral load measurement.
We agree! You need delta delta Ct to get a load. That requires an internal control like RNaseP. CD protocol doesn't have internal control primers.
A single Ct is Not viral load measurement.
We agree! You need delta delta Ct to get a load. That requires an internal control like RNaseP. CD protocol doesn't have internal control primers.
11:20
Re-emphasizes the need for proper controls.
We agree here. As does the body of work surrounding MIQE.
Re-emphasizes the need for proper controls.
We agree here. As does the body of work surrounding MIQE.
15:00 mins in
Dr. Bustin claims SARs is single stranded RNA.
SARs is double stranded RNA.
This isn't relevant to the CD discussion but felt the need to clarify as we agree the colder RT temperatures are where a lot of the mis-priming happens.
Dr. Bustin claims SARs is single stranded RNA.
SARs is double stranded RNA.
This isn't relevant to the CD discussion but felt the need to clarify as we agree the colder RT temperatures are where a lot of the mis-priming happens.
19:00
Late Cqs are a problem.
We fully agree.
This is a good place to reflect on LODs mentioned in MIQE guidelines.
Late Cqs are a problem.
We fully agree.
This is a good place to reflect on LODs mentioned in MIQE guidelines.
21:00
Primer Dimers
"Can reduce the sensitivity of the assay"
We agree. Primer dimers can also create FPs as shown in our Addendum. This is less common on Taqman assays but not impossible and demonstrated in multiple peer reviewed papers using CD in our Addendum. Jaeger et al.
Primer Dimers
"Can reduce the sensitivity of the assay"
We agree. Primer dimers can also create FPs as shown in our Addendum. This is less common on Taqman assays but not impossible and demonstrated in multiple peer reviewed papers using CD in our Addendum. Jaeger et al.
23:00
"Primers shouldn’t bind to each other and they shouldn’t have mistakes in them (admits one exists in CD), and they shouldn’t bind to other viruses.”
All of these deficits exist in the CD primers.
This is more thoroughly dissected in the Addendum and perhaps he missed this
"Primers shouldn’t bind to each other and they shouldn’t have mistakes in them (admits one exists in CD), and they shouldn’t bind to other viruses.”
All of these deficits exist in the CD primers.
This is more thoroughly dissected in the Addendum and perhaps he missed this
"I haven’t looked at the sequences to see if they bind to each other”
This is shocking.
If you haven't done this and you reviewed the retraction request which points this out… what did you actually do for the review?
Is "Utterly Absurd" warranted if you admit this?
This is shocking.
If you haven't done this and you reviewed the retraction request which points this out… what did you actually do for the review?
Is "Utterly Absurd" warranted if you admit this?
If he is the reviewer of our retraction request, He should look must closer here.
The primers do bind to each other and if you admit to not having looked at this and its the crux of the issue, then the community is not going to be content with said review.
The primers do bind to each other and if you admit to not having looked at this and its the crux of the issue, then the community is not going to be content with said review.
Speaks to the the need to use in-silico analysis and scan primers so they don't hit other sequences or hit themselves. Offers software to do this.
We agree with him and we pointed this out in our retraction request that its pretty clear CD didn't do this.
We agree with him and we pointed this out in our retraction request that its pretty clear CD didn't do this.
26:00
Discussion on Cases.
Second qPCR test or RAT test would help define cases that are actually infectious.
We Agree with his logic here. There is a large window of qPCR +ve when the patient is not infectious. These are clinical false positives as long as PCR calls 'cases'.
Discussion on Cases.
Second qPCR test or RAT test would help define cases that are actually infectious.
We Agree with his logic here. There is a large window of qPCR +ve when the patient is not infectious. These are clinical false positives as long as PCR calls 'cases'.
30:00
Probe discussion- doesn’t give signal if properly designed.
We demonstrate the are not properly designed in the addendum. RdRp probe hairpins and homodimers.
Probe discussion- doesn’t give signal if properly designed.
We demonstrate the are not properly designed in the addendum. RdRp probe hairpins and homodimers.
37:00
2 or more genes. We agree!
What happens when its 2 genes and ...
1) is the non-specific E-gene that hits other viruses
2) is the insensitive RdRp gene that dimerizes and
3) is the N gene that authors admit doesn’t work.
You don't have 2 targets. You have 3 broken ones.
2 or more genes. We agree!
What happens when its 2 genes and ...
1) is the non-specific E-gene that hits other viruses
2) is the insensitive RdRp gene that dimerizes and
3) is the N gene that authors admit doesn’t work.
You don't have 2 targets. You have 3 broken ones.
39:20
His opinion on the CD retraction request-
"Utter Discrace..
Absolute disgrace"
Mentions he is one of the @Eurosurveillanc reviewers for the retraction request.
That is shocking to hear in an interview where he admits to not looking very closely at the CD primers???
His opinion on the CD retraction request-
"Utter Discrace..
Absolute disgrace"
Mentions he is one of the @Eurosurveillanc reviewers for the retraction request.
That is shocking to hear in an interview where he admits to not looking very closely at the CD primers???
44:00-
Admits they were not perfect primers and had mistakes.
This is a good time to think about 27hr peer review that had conflicts of interest.
Admits they were not perfect primers and had mistakes.
This is a good time to think about 27hr peer review that had conflicts of interest.
46:00
Again, admits they have mistakes but other 2 sets were OK..
No they were not!
N gene didn't work.
E gene was not specific
RdRp has dimers and sensitivity issues.
NCBI accession numbers on the targets the test was validated on only have RdRp sequence?
Again, admits they have mistakes but other 2 sets were OK..
No they were not!
N gene didn't work.
E gene was not specific
RdRp has dimers and sensitivity issues.
NCBI accession numbers on the targets the test was validated on only have RdRp sequence?
47:45
Mentions PCR should never get a FP if you have designed correctly and have internal controls.
We agree. However the CD protocol have design flaws evident to those who do look at them and they have no Internal controls (ICs).
A good read on ICs. https://pubmed.ncbi.nlm.nih.gov/33131699/
Mentions PCR should never get a FP if you have designed correctly and have internal controls.
We agree. However the CD protocol have design flaws evident to those who do look at them and they have no Internal controls (ICs).
A good read on ICs. https://pubmed.ncbi.nlm.nih.gov/33131699/
53:00
Dr. Bustin displays a pro-lockdown position being correlated with better C19 outcomes which is unsupported by the data. There is no correlation with qPCR testing volumes and better C19 outcomes, despite $40B+ C19 PCR industry and massive economic destruction from quarantine
Dr. Bustin displays a pro-lockdown position being correlated with better C19 outcomes which is unsupported by the data. There is no correlation with qPCR testing volumes and better C19 outcomes, despite $40B+ C19 PCR industry and massive economic destruction from quarantine
Dr. Bustin does have an interest in qPCR testing for C19. While I use PCR in a different industry, I have no C19 qPCR financial interests.
Dr. Bustin is quite qualified to weigh in on this topic given his seminal work on MIQE.
I hope his pro-lockdown positions can be tabled and provide him with enough neutrality to look at our Addendum. He will see, this assay would be condemned by many statements in MIQE.
I hope his pro-lockdown positions can be tabled and provide him with enough neutrality to look at our Addendum. He will see, this assay would be condemned by many statements in MIQE.
It is quite possible, he only saw our Nov. 27th retraction request lack much of this detail. But to go on a live cast and admit to not having looked closely at these primers and in the same breath having performed a serious review of our concerns...Looks like lockdown bias.
