I finally found the cause of my qPCR virus tests failing for a recent cold virus I knew we had. Through sequencing, I learned my virus happened to have a novel C->T mutation right at the 3’ binding site of the “HRV Ma” primer I use!

I’m seeing better (but, to be honest, still unsatisfying) results from the primer labeled “ENT” which is described in https://www.nature.com/articles/srep34855. qPCR plot, green lines HRV, red ENT.

The different lines in the above plot are trying different primer/probe concentrations. I say the ENT results are unsatisfying because I really had to bump up my primer concentration (500nM to 1.5µM final), the ∆RFU is still too low (600 vs 2000), and plot misshapen.