This might be interesting..

Enferm Infecc Microbiol Clin. 2018;36(6):332–335

The fluorescence intensity is target-specific. This is the case for SYBR-Green and even more so for sequence-specific probes. Further, the threshold used for a target is arbitrary. This is why one needs an external standard to make such measurements comparable (even for the same target between different runs/plates).

This is very similar to e.g. ELISA measurements. You wouldn't expect to get the same OD reading when using different antibodies against a protein, or when comparing readings using the same antibody between plates.

Well, that tells me that the different primer pairs have slightly different efficiencies and/or different detection methods. Higher efficiency means you can have a lower threshold for significance. If some are using probes and other are using SYBR, then you would expect different Ct values.

The root cause is technical, not biological.

Thanks Katie A S Burnette , Jochen Wilhelm and Talha Bin Emran for your answers which all make sense

My thoughts about it is that

-it could be due to the abundance of the specific targets , the more the earlier being detected

- or possibly something to do with the assay competitivity nature.

Hi,

While optimization , preferential amplification of the target which give precise results (for example N gene, RdRp gene) will be considered as major factor over the supportive target (for example E gene). This can be achieved by altering or adjusting the gene specific primers and probes, melting temperature etc.. Even if you give exact copies of each gene separately, you will not get same Ct value.

Individuals who develop own multiplex PCR or real time PCR often use this concept. Hence, the sensitivity obtained for the supportive target is often inferior than the major target. However, as a whole assay such adjustments should not alters the sensitivity hugely.

hi Narayanan Kalyanaraman

Thanks for your answer. it really make sense to me.