Hi! I'm currently having issues with qPCR assays on viral specific primers. The general assay and chemicals are commony run in our lab and always shows real good results with efficiencies between 96-100%. Now, working with virus-specific primers that somebody designed I'm running into issues.I quantified the plasmids for the Std curves using Qubit and subsequently made 10 fold dilutions (10^8 copies 10 fold down to 10^1 copies as well as 5 and 2 copies). The Std curves look fine with R2 of 0.998 but the slope and subsequently y intercept are too high bringing efficiencies down to 88%.For one of my primer pairs the Std curve looks good, I'm able to detect everything down to 5 copies but efficiencies are low (slope too high with 3.66 and 47 intercept). Delta Ct values alternate between 4.0 (10^8-10^7), 3.64 (10^7-10^6), 3.53 (10^6-10^5), 4.38 (10^5-10^4), 3.24 (10^4-10^3), 3.5 (10^3-10^2), 3.18 (10^2-10^1) and 2.35 (10^1- 5 copies).Another primer pair did not amplify at 60C but showed best amplification at 50C. I ran qPCR at 55C then but it also shows low efficiency (due to high slope) but additionally has the problem of not showing any detection below 10^3 copies.Any input would be highly appreciated!Thanks!