PCR Controls

A very Positive, Negative.

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The content of this post will be a Substack only post as the results need the long form explanation behind them to understand them.

So far with the project, every experiment we have conducted has proved our hypothesis correct. In the 12 experiments in Cell Culture Controls with no added sample the CRO measured CPE in ALL 90+ cultures, measured with objective verification of the COUNTESS cell viability counter as well as observed CPE morphology in the Cell Line supposed to indicate a “virus”(Cell rounding,clumping, syncytia, lifting, floating). This was within the time frames and amounts according to the American Society of Microbiology.

We then managed to identify on our first attempt in just 6 TEM photographs, particles of the same shape, size, coating and inclusion to the CDC documented images and descriptions of “Sars Cov 2”, “Measles” and “Hiv”.

But we do have some results that are yet to be published that I think, with explanation are actually very revealing.

I made a very rookie error and will totally hold my hands up about this (but maybe you will come to see that it might have been a stroke of luck) but I engaged a CRO to do RT-qPCR testing of 5 of our controls cultures, but told the CSO (Chief Scientific Officer) what the aim of the project was. I.e I let the person in charge of carrying out the experiment KNOW that the cultures couldn’t possibly contain a virus.

My alarm bells should really have rung when I was ushered to have a 45 minute interview with the CEO of the Genetics Lab asking me who I was and what I was trying to achieve. This is an entirely unorthodox approach to Contract work within the Scientific industry, with all of the other work done with CROs when blind to the aims of the project they have asked for bare minimum questions, usually refined to simple administrative or logistical enquiries.

We signed off on doing an initial run of just 5 samples to see where we were and with an aim to test a full 96 well plate. We elected to use what is considered to be the most accurate test for “RNA Viruses” of PCR the Reverse Transcription Quantitive PCR or RT-qPCR for short. We also elected to use what is considered to be the most accurate 3 probe Primers for “Sars Cov 2”.

Despite choosing the most accurate form of PCR test available in the manual for the primer kit we used it STILL gives a disclaimer that these tests are NOT to be used for diagnostic or therapeutic purposes.

Even a positive test from the most accurate form of PCR should NOT be treated medically or assumed that this is the cause of disease. Right here we already have some pretty damning legal evidence for the way people were treated in hospitals based on these tests.

Also, a positive from the tests does NOT actually mean that the “virus”is present, you must confirm with Whole Genome Sequencing before you can say for certain. This is why we are focusing on Whole Genome Sequencing controls currently, as being the end of the line for all of the “genetics evidence”.

When having the meeting with both the CSO and CEO of the genetics lab, the topic of primer selection came up. Because they were both privy to the knowledge that there couldn’t possibly have been a “virus” in the samples, this seemed to be a large hurdle of choosing what to look for.

I suggested that there must be a non-specific primer used for when you didn’t know what was in the cultures. According to the text books you should have a fair indication from symptoms of the patient the sample was taken from. But this surely can’t tell you for instance between all of the millions of different variants of Influenza and Coronavirus.

So I assumed there must be some sort of generic primer that could tell you the ball park any “virus”in the dish was in… i.e have you got an Influenza or Coronavirus in the culture to then narrow it down. Apparently not only did this not exist, it was very difficult for the CSO to even comprehend what I was asking for. LOL. It seems as if you have to be a fortune teller if working in the sector of Genetics. You have to KNOW what virus you are looking for BEFORE the test, so you choose the specific primers only. I found this in and of itself very revealing as it shows the whole process is through predictions and molding of input materials.

Next up on the very surprising list of information came also through this meeting. I had been working with a seasoned geneticist that is part of the project talking through all of the ins and outs of the PCR process from extracting the “RNA”through to primer design. They had offered to make the primers to test the cultures with. After some deliberation we decided not to go this route as it could be construed that we were gaming/biasing the system.

As part of these preliminary discussions our geneticist had done a couple of dry runs of the PCR themselves and pulled up a few oddities. One being that one channel of the PCR registered positive where as another didn’t amplify at all leading to a very strange curve and output.

Here is a part of the manual of channel calibration.

On interview with the independent genetics lab I put this scenario to them and asked if they knew what the problem with this might be. The almost instantaneous answer back was “Oh that’ll be the internal thresholds need adjusting”. As I had only ever heard of the term Threshold in reference to Cycle Threshold (CT) before I inquired back ; “Oh they need to adjust the CT?” They replied back “No that is after amplification, this is a threshold for the machines sensitivity for background noise”.

I thought this was very odd that there seemed to be hidden parameters which could dial up or down the sensitivity of the machine. A little Googling later and Lo and Behold I found this thing called Baseline Correction. It is an internal threshold to adjust for “Background Noise” but seems it can be dialed up or down to affect sensitivity right across all channels. In Layman’s Terms… it is adjusting how sensitive the camera is for picking up the flashing lights from the Fluorescent Dye they intentionally put in.

This means that the “CT”value is effectively altered up or down depending on this internal threshold. Again in simplistic terms it means that a CT value of say 35 can mean anything from 20 to 50 cycles relative depending on this Baseline Correction.

This was just from the Baseline Correction alterations given in the manual above for setup with a value of up to 15 cycles different. Is it possible to go even higher? My instincts would tell me so.

Positive? Control

As part of the qPCR test we have two controls. One is the “negative control”which is just the primers and nuclease free water. This obviously doesn’t constitute a proper negative control as it contains none of the same ingredients of the sample - “the viral RNA/DNA” but nevertheless we won’t focus on that for the moment. The other control is the Positive Control, meant to consistently give a positive and the strongest positive as a benchmark because it is meant to contain essentially purified Independent Variable or in this case purified Nucleotide Sequences for “Sars Cov 2”.

What do they use for the Positive Control? A “purified virus titre”from culture right ? Well no. They mix together over 200… yes TWO HUNDRED different chemicals in over 50 methodological procedures, NONE of which are controlled at any point to create what they call an Oligonucleotide. This is meant to be PURE nucleotide sequence of exactly the nucleotide sequence you are looking for in PCR.

So if you put a PURE nucleotide sequence into a PCR machine with Primers that ONLY bind to that nucleotide sequence how many cycles do you think it should take to amplify? My guess and I would expect that is a rational guess would be 1 cycle? Maybe if we are being generous say sub 5 cycles? Well… I was wrong….very wrong.

These EXACT nucleotide sequences can take up to 27!! cycles to amplify and still be called a legitimate experiment.

One last thing to clear up before we get to the results of the PCR test on our cultures. The Cycle Threshold or CT is the point at which the curve of the sample being tested breaks to being exponential in amplitude. This usually is potted on the PCR graphical display and usually comes with data columns to show the exact Cycle Values that the curve amplified at usually to at least one decimal place.

The Results

So we received back the test results and the Genetics Lab said that unfortunately the samples were all negative and that they recommended we get them whole genome sequenced to find out what “virus”was in the culture that caused our observed CPE.

This was a bit stomach churning to take, we had received “positive”confirmation on EVERY try for the other areas of this. It doesn’t mean that our hypothesis was falsified as samples CAN be negative. If it was just the primers affecting it then you would expect to ONLY see positive results or couldn’t possibly see mixed results from the same batch with the same volumes of primers in. So there MUST be something in the sample they are registering.

*See my Article DIY Control for some thoughts*

Initially they just sent through verbally what the results were, it was only when I followed up that any of the graphical data was given to us , which was a bit of a give away. This is what was given to us:

To talk you through this as NONE of it was labelled. The lines that are all amplifying are the “Positive Control”. There was no Data that came with this i.e exact figures of CT values and there was no CT Threshold line marking where this data would have indicated from the plot.

So with the confirmation of the geneticist that works with the Project who confirmed everything I will tell you, we plotted on the Cycle Threshold for the Positive Control.

That indicates that the Positive Control amplified at 35 CYCLES!! If that were a sample, most labs would call that a redo. But categorically 100% according to the manual for the primers used, this experiment has FAILED as the positive control amplified over 27 Cycles.

Also to note is that the Line for the Positive Control is quite “Lazy”, it usually takes only 1 or 2 CT values to reach exponential as this is meant to be an incredibly strong benchmark readout. In this case the Positive control steadily rises from around 20 cycles until finally breaking exponential at 35… a whole 15 cycles later.

So it would have to be speculation but there must be a reason for this test failing by pushing the Positive Control to amplify at such high CT values. My Speculation would be that because the genetics lab were so “Au Fait”with Baseline Correction and they were so scared of bringing back a positive result on a culture they KNEW couldn’t possibly contain a “Virus”, they intentionally whacked up these baselines so that NOTHING could test positive other than the Positive Control.

If you look at it this way it would be IMPOSSIBLE for anything to amplify if the PURE sample of Nucleotides weren’t being tested. For a PC to amplify at 35 cycles which would usually plot at say 17 (fairly regular PC value, still high IMO tho), even a VERY strong positive sample would be low 20s CT. Given this, to me, obvious Baseline Correction would amplify at 40+ Cycles and so would never plot.

Conclusion

There we have it, a Negative result, but some extremely useful information learned from this process. Maybe more useful than receiving back a positive as we have potentially found a huge area of manipulation in these tests that were otherwise unnoticed.

1. The CT value may not be the CT value…. lol… it very well maybe completely different given the Baseline Correction for the thermocycler.

2. Positive Controls aren’t always that “Positive”, note that they NEVER give you the Positive Control value as a benchmark when hearing PCR test results, this area alone needs a lot of scrutiny.

3. The Importance of BLINDING in this process, the labs CANNOT have any idea of what is or isn’t in the culture, especially when it comes to our mythical “viruses”.

4. It seems that it is totally possible to manipulate and defraud negative OR positive results from the machine settings alone.

5. Even if all had gone to plan, the get out clauses were always in place with PCR not being for “diagnostic or therapeutic”use and also not necessarily being accurate and needing confirmation in Whole Genome Sequencing.

We will no doubt return to do some BLINDED PCR tests as part of the project, but for now are focused on the end of the line with Whole Genome Sequencing….. watch this space.