I usually do RT-PCR test for covid-19. Curves for Patients and Positive CTRL mostly appeared at 20 to 25 Ct. Sometimes, there are some curves that emerge at the last 5 cycles. Some argue that they are probably primer dimer and should not interpret as positive. How primer dimers would appear on the amplification graph?
$\begingroup$ I assume you do a proper negative control, right? $\endgroup$– Chris ♦Jun 3, 2021 at 6:26
$\begingroup$ Negative CTRL is double-deionized water. $\endgroup$– Arash SalehiJun 3, 2021 at 7:01
1$\begingroup$ If you're testing for diagnostic purposes, you should probably be using a kit with sequence-specific fluorescent probes, not an intercalating dye like SYBR. Primer-dimers won't show up as florescence in a probe based PCR, but they can reduce reaction efficiency, leading to later detection of your target. You should also consider running a negative specimen control, along with your water control, to screen for possible contamination events during extraction or sample prep. $\endgroup$– MikeyCJun 4, 2021 at 21:50
This is a bit complicated from far away, but I will share a few thoughts on it. The occurence of primer dimers depends on the design of the primers (sometimes they are inevitable due to sequence limitations), but then they also show up in the negative controls.
If they are not present in the negative controls (meaning: never), I would not expect them to be present in the test reactions either. It is possible that you are getting very late unspecific DNA amplification from other sequences due to degrading etc. but this is also unlikely.
You could do a melting curve analysis to see what your product looks like. A pure product (even in the late phase of the PCR) should show up as a sharp and clearly defined peak, whereas primer dimers (and also the products of unspecific DNA amplification) have generally a much lower melting temperature and give a much broader peak. The figure (from here) illustrates this nicely.
You could also run your samples on a gel after the PCR and see what the product looks like. Product gives nice and clear bands, while byproducts are smaller and more diffuse.
It is not unlikely that you either tested people who have gone through an infection with SARS-CoV-2 and have only some virus left in their body or that the sample comes from people very early in their infection, when the detectable amount of virus has just gone over the threshold for detection. Assuming that the samples have been correctly taken (which is another possibility for error), I would collect a new sample from these individuals and test them again.
2$\begingroup$ I would just add to this that most commercial PCR tests for Covid diagnosis use sequence-specific probes for detection, not intercalating dye. With a probe assay, you should not see fluorescence increase from primer dimers alone, because the probe should still have nothing to hybridize with. That said, most commercial kits also have a pre-defined cutoff cycle for amplification to be considered positive. The utility of these cutoffs can be debated, but if you're testing for diagnostic purposes, I would not recommend deviating from those specifications. $\endgroup$– MikeyCJun 4, 2021 at 20:43