Request for information from anons educated in PCR test.
🧐 Research Wanted 🤔
My wife is required to submit to weekly testing for the coof. I asked the testing facility what Cycle Threshold they use for their tests. Here is the response:
**Our test does not use cycle thresholds (Ct). Ct is used for a qPCR test method, whereas we perform rRT-PCR. Our detection is given by flourescently tagged molecules that generate Median Fluorescent Intensity (MFI) based on the viral load of the collected sample. We test for multiple genes within the SARS-CoV-2 genome and have a different threshold for each. I hope that answers your question. **
I 'think' I understand but perhaps there is an anon in here that can give a brief explanation regarding accuracy etc.
I am certified molecular technologist, I perform rRT-PCR,
They danced around your question, I have seen a test that is <40 Ct cutoff for SARS and <38 Ct cutoff for Internal control
All these tests should test multiple gene targets, and each may have a predetermined (supposedly through validation processes) to be acceptable Ct for that gene,
The <40 act is a pretty iron clad rule as the upper limit just do to the nature of the reaction which involves multiple cycles of heating and cooling so each time the machine heats and cools the reaction plate that is one cycle and this occurs repeatedly over many cycles like 45 cycles for example
The threshold for each test is actually kind of an arbitrary value that is set independently in the analysis process, it’s supposed to be determined by comparing the negative controls to the patient samples, this establishes a line or threshold
When a gene target is present in sample this causes amplification or copying of the dna in the sample and this increases fluoresce which the machines detect
So where does cycle threshold come from? It’s the cycle at which point the fluorescent signal from a target in a sample crosses the threshold this is generally represented by an exponential increase in signal
Graphically exponential increases can be thought of as a vertical line so raising the threshold shouldn’t alter Ct of a true strong amplification signal
The issue is when you have poor or weak signals that raising or lowering the threshold can have a drastic effect
Additionally the process of heating and cooling over repetitive cycles is highly stressful on the enzymes that perform the reactions, eventually they make mistakes and you get amplification of molecules that are not true signals and this is where raising the Ct as the WHO and CDC demanded causes issues because after 40 it’s likely background signal but it can be made to cross the threshold so it’s being counted as a positive because they raised the cutoff on whats an acceptable value range
So in summary she told you part truths and danced around the question which was what their ct testing cut off is for each gene target
You should follow up and ask:
If you know where they got accredited from Then if they give you a hard time say you’re going to report them to CLIA, COLA, or CAP or whatever company handled their accreditation say you’re going to file a complaint and report because you do not feel confident they are keeping up with the LAW: CLIA
And if they answer your questions it’ll probably at least at face value appear to be ok, it’s not hard to lie and give acceptable ranges even if they don’t use them...
good luck hope i was able to help in some way