I already used the Pan-papilloma primers (FAP59/64) for detecting camel papilloma virus in skin nodules that worked perfect. Now I like to develop a qPCR assay, but as papilloma viruses are genetically heterogeneous and you cannot easily find an area within the genome having conserved sequences you must use degenerate primers. However, I have no idea if qPCR can work with generic primers. Any help?
Hi,
We routinely use degenerate primers and probes for qPCR assays.
If your conventional PCR works well, then there should be no problem using a SybrGreen PCR provided that your amplicon is not too large. Just be sure to run a melt curve for primer dimer (will artificially increase RFU) and unspecific amplification. If these two factors are negative (i.e. specific amp with no primer dimer), and you have a good efficiency then you can quantitate with standards.
Alternatively, you can design degenerate probes to form hybridization/hydrolysis PCR assay to increase the accuracy of your RFU data. However designing good probes is a bit of a guessing game when it comes to heterogeneous regions.
My first step: check the literature for previous validated qPCR assay systems to prevent re-inventing the wheel.
Good luck and hope it helps.
Dear Chris,
Many thanks I really appreciate your answer which I found very useful and promising. The PCR product when using the published Pan Papilloma primers measures 478 bp in the gel-based assay, a size not suitable for qPCR. Therefore, I designed using an online free software a set of primers and probe, all degenerate. For the probe the sequence look like: aaytggsagatwggmwtgcarccm.If the qPCR in principle can work with degenerate primers then my worry is possibility of synthesis and labeling of the probe by commercial companies in term of possibility and cost. Any idea?. Search of literature revealed no qPCR for papilloma virus. I will be happy if someone correct me and mention to me the needed article.
The pair described here might be suitable for Sybr green detection, since product is only 150 bp.
Dear Abdelmalik,
You are more than welcome. Shortly after replying to your question I was searching through some cases and found that I have to test for papilloma by TaqMan PCR.
You are correct that the size of the product is large for qPCR, but it could still work. It is unlikely that quantification will be possible but I would certainly still perform a SybrGreen assay before ruling this out. There is a reasonable chance that you can have a qualitative assay.
Regarding the probe, it can be a bit of a guessing game as many factors can affect if it will work or not. They are expensive but also last a long time. The manufacturer should have no problem to generate this for you, and they are responsible for the Q.C of their product. Regarding a 478bp TaqMan PCR, I would only attempt if there was nothing else.
In my opinion, I would use your current system with a SybrGreen mix, run a melt curve and run the gel. If you decide to do this I would perform 10-fold dilutions of the positive control to calculate the efficiency to understand how well the system is working.
Good Luck, I hope it works!
Thanks David for the paper, this 150bp is much better suited for qPCR. I will investigate for my TaqMan system
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