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I am from IT/ Engineering background and have some confusion in RT-PCR. So far my understanding we extract RNA from test specimens and transcribed it into complementary DNA (cDNA), and inside PCR DNA double helix is separated. Then primer a short nucleic acid sequence that provides a starting point for DNA synthesis with the target sequence and multiples the target DNA.

Since a higher cp value indicates a negative result but why negative results have cp value? Why it cross the threshold when it does have any target sequence (viral DNA)? Fluorescence is the measure of target DNA right??

In extraction, RNA is extracted does only COVID viral RNA is deducted? What happens to other viral and human RNA?

I have seen result were we have low cp value and test result is negative, the reason for this was because curve do not have Sigmoid function. I need more explanation on it.

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  • $\begingroup$ I do research on these tests for a living, and I've never found a good explanation for why some PCR reactions produce these linear amplification artifacts, even amongst my clinical diagnostics colleagues. You could try running the product of them out on an agarose gel to see if you see the expected band (or bands). But I agree with the Chris's answer that, if you're unsure about a result because of a strange shape to the amplification curve, I would re-test the sample, probably doing a fresh RNA extraction, rather than simply making a negative determination. $\endgroup$
    – MikeyC
    May 20 at 14:14
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  1. PCR is highly specific (meaning it amplifies only the DNA the primers bind to), so the results in general are good. But: Due to the exponential nature of amplification, even very small mispairings can lead to the amplification of a very small amount of DNA and subsequently to a fluorescent signal. There are basically two ways of getting this amplification (except from the desired one): Contaminations and primer self-amplification. The first one should be clear, the second one happens when primers are able to bind to themself (or the complimentary one). This usually shows up late in the PCR, which is the reason why you assign a Ct-value to it after which you do not expect any meaningful amplification result anymore.

  2. Depending on the method, you extract all RNA from your sample and translate it into cDNA. However, for the amplification in the PCR you need primers that fit to this cDNA. And since these primers have been proven to be highly specific (meaning: they bind absolutely no other sequence, not even from closely related viral species) we can be sure that any signal which is about the criteria mentioned in 1. comes from viral RNA and shows an infection. See the reference for one of the often used PCR tests.

  3. Without seeing the curve, I cannot say anything specific about it. In general a real-time PCR curve follows a sigmoid shape. This gives some information about the way the reaction progressed over time. If it looks differently, something may have either happened to the reaction or to the sample. It may contain a PCR inhibitor, the sample can be contaminated or something else. I wouldn't trust such a result and repeat it with a new sample.

Reference

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

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    $\begingroup$ Just a side comment, but for diagnostic purposes it's often not the case that All RNA is reverse transcribed into cDNA. Most nucleic acid tests for SARS-COV2 (and other RNA pathogens) use a target specific RT step, so that only regions containing the PCR targets are reverse transcribed, helping improve specificity and reducing RT bias. Since the reverse primer for the PCR will generally perform this function well, these tests often use a 1-pot (or 1-step) RT-PCR, which is faster and also slightly more sensitive since you don't dilute the cDNA when adding it to the PCR mix. $\endgroup$
    – MikeyC
    May 20 at 14:04
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While @Chris's answer is correct, there is a complication that comes into it:

The principle behind quantitative PCR (qPCR) is that you have a reaction which, if your reaction is 100% efficient, doubles the number of DNA strands produced at each cycle. In theory, you can start from one strand and get 2, then 4, then 8 in a 2n style equation, where n is the cycle number.

However, in reality this rarely happens, most reactions are not 100% efficient; generally in the 90% range is considered acceptable, so if you do the maths, it doesn't quite work out at a doubling each cycle.

The qPCR tests run on COVID-19 samples are generally run out to 45 cycles, which if you work out the maths, means that if a sample is showing some amplification at this point, then there is less than 1 copy of the RNA (actually cDNA) in each reaction, so the signal is likely to be non-specific amplification or spurious signals from other products in the reaction, such as primer-dimers (shouldn't be the case in these tests though, they use a system that all-but eliminates this problem).

Non-specific amplification can be detected by looking at the reaction and comparing the melt-curves of the products of the PCR. Specific products will have melt-curves that meet certain criteria (all with a peak at the same point), while non-specific products will have different melting points/curves.

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