Thread by @michaelmina_lab

THREAD: Maybe you’ve heard about Ct values in the qPCR test and viral loads.

So what’s a Ct value, why is higher Ct a lower viral load, what’s it have to do with #COVID19 , and why do we care?

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Ct stands for 'Cycle Threshold'

In qPCR tests, when the virus RNA enters the test, to detect whether virus RNA exists, it has to get amplified to be seen by the machine, like a ‘zoom’ feature.

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The qPCR machine has detectors that detect fluorescent light.

Importantly, in qPCR, fluorescent molecules bind RNA (actually DNA, but for clarity, I’ll keep calling it RNA since it’s RNA in the virus and people know RNA now - but really the RNA becomes DNA in the test).

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So if there is viral RNA, the RNA will fluoresce and can be detected by the detectors – small cameras measuring fluorescence.

The detectors are good, but to really ‘see’ the RNA, the signal has to get brighter – like zooming in on your computer.

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To “zoom” in, the PCR machine ‘cycles’. With each individual cycle, if the virus RNA is there, the RNA will double, and the fluorescent molecules that stick to the RNA will get twice as bright.

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So if there is 1 RNA molecule, after 1 cycle, there will be 2 RNAs and the signal is 2x as bright. Another cycle makes 4 RNAs and the signal is 4x as bright compared to starting.

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The Ct or ‘Cycle Threshold’ is therefore the number of cycles it takes to make the signal bright enough for the detector to see it… If you have to go through a lot (i.e. 30+) of cycles to see it, then you started with a small amount of virus RNA

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If you have to go through only a small number of cycles (i.e. 8-15), then you started with a lot of viral RNA.

Every increase of ~3 cycles needed to see it means a 10x lower viral load. Add 10 cycles = 1000x lower

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If sample A has Ct = 20 and sample B has Ct value = 23, then A is ~10x higher viral load than B.

If sample A has Ct = 37 and sample B has Ct value = 27, then A is 1000x lower viral load than B.

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So, again, Ct is like zooming in on your computer screen. If you have to zoom a lot, then the thing was small to start with. If you have to zoom a little, then the thing was big to start. In PCR, the ‘thing’ is the starting amount of virus.

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To sum, Ct is inversely proportional to virus load.

It is the # of cycles or “zooms” needed to see the virus RNA. If many cycles/zooms needed, it means low viral load input

If few cycle/zooms to detect means you high viral load input

each +3.3 cycles = 10x lower virus

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