Virus specific qPCR

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Lisa Sudek
Lisa Sudek's picture
Virus specific qPCR

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!

Ivan Delgado
Ivan Delgado's picture
qPCR assays not working

Hi Lisa,It is hard to know where to begin with your situation. You are saying that the "general assay" shows good results but the "virus-specific" primers that "somebody designed" are not working well. Because I do not know what is the difference between your "general assay" and your "virus specific" assay, my best guess is that your "virus-specific" assay is not working or was not well designed. It is also possible that your virus DNA is not good.Another thing you may want to look into, considering that your slope is too high, is that the DNAs you are using may not be within a good range for this assay. In other words if you are using too much or too little DNA the assay is simply not going to give you a good slope. Get rid of some of your too high/too low dilutions, or instead of performing 10-fold dilutions use 2-fold or 5-fold dilutions. This way you still have enough points in your standard curve but are not asking too much from your assay by trying to amplify from too much or too little DNA.Hope this helps. Ivan

Lisa Sudek
Lisa Sudek's picture
qPCR assays not working

Hi Ivan. Thanks! That was real helpful. I will try different dilutions for Stds first and see where that will get us.Lisa

Nikail Collins
Nikail Collins's picture
primer design

Going back to look at the primer/probe design might be useful.  Maybe, a comparison of the primers in assay that works well vs not so well in relation to reference strains of the target virus.