I am running qPCR assays for porcine parvo virus. The assay is running fine except for the fact that my negative control wells show an amplification curve every time I run the assay. The Ct values of the negative control plates are almost similar, implying that a reagent used in the assay is contaminated with the amplicon DNA. I have bleached the working area and used DNA Zap, bleached my pipettors, thrown away all reagents and used new ones for every assay, tried a different working area and every possible thing I can think of. But, the contamination persists. Can someone suggest something that I may have missed or have been doing wrong ? Thank you.
Did you check the melt curves? This sounds like primer dimer/secondary structure problem.
If you have primer-dimer structure the Tm might be around 72-75 deg, Then you will see double peaks after melting curve analysis.
The qPCR assay is probe based and NOT SYBR green based. Is the primer-dimer structure relavant in probe based assays ??
I understand. I won’t work. You might check this running real-time PCR with Sybr Green without Any Taqman probe.
Did you do an electrophoresis to see if the NTC contamination has the same size then you virus amplicon?
No I have not run a gel, but that's a great idea. Thanks. I'll let you know what I find
If it is the same size, you could also send it to sequencing. If it appears that your reagents are contaminated, here a useful paper: Champlot et al. 2010 (An efficient multistrategy DNA decontamination procedure of PCR reagents for hypersensitive PCR applications.) And sure, let me know what you find, good luck!
I have suddenly had the same issue. The NTC were coming up at the same ct as my target which is in low abundance anyway, giving me false positive results. It ended up being probe degredation from a recently purchased probe (which is now getting replaced). This can happen at the supplier end if incorrect bleaching occurs and can occur if you don't aliquot your probe out and only freeze thaw what you need.
So I ran my NTC wells on a gel and the band size is similar to the viral amplicon. Since then, I have bleached and DNA Zapped my workstation and tried to run only NTC wells with no DNA on the entire plate. The contamination seems to have subsided, but not completely gone (10% of the wells were contaminated). I asked a colleague to run the same assay and her NTC wells have no contamination at all and the assay is running fine. This puts me in a even more confusing position.
Is there a possibility of your pipettes coming in contact with amplified product?
You can add dUTP to the qPCR mix which would help in preventing carry-over contamination to the next run. The subsequent qPCR mix would have to be treated with Uracil DNA glycosylase. Some firms offer this option in their master mix.
I saw that some time ago. At the end, it was a problem with the probe. We had amplification on the NTC, repeated everything after cleaning all the surface, pipettes with DNA Zap and still had that "contamination". Primers were redesigned and still the same amplification profile on the NTC. Finally, the probe was redesigned and everything solved :). Good luck!!
Thank you for all the responses. I have tried decontaminating everything and am now in the process of ordering new primers and probes.
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