Your standards look nice, but you are right, the experimental samples are unsatisfactory. I would worry that there is something in the sample that inhibits the reaction. When I have done qPCR on PyVs (non-clinical samples however), I tried to stick as close to the Murata method as possible. The viral DNA should be quite stable if you need to alter your harvesting/clean-up methods. How do you harvest the DNA for this reaction?

(J Virol Methods. 2009 Dec;162(1-2):236-44. Epub 2009 Sep 1.

A quantitative PCR assay for SV40 neutralization adaptable for high-throughput applications.)

Hi Anna, I would also try to dilute some more your samples, I had a problem like yours when I was working with HIV. When I diluted the samples 1:10, 1:100 I had better results.

It could be that the PCR is getting inhibited by excess human DNA in the samples. have you checked the quality of the DNA? Also what method are you using for DNA extraction? You could check the 260/230 ratios to rule out inhibitors like chaotropic salts and/or phenol contamination

Hello Anna,

I have just gone through your picture you have attached herein. Don't you think that your threshold value is too low. I think you set it manually and I doubt that this threshold value is machine-default. Am I right? Your threshold value can't be so low that it incorporates even the background noise -- as I can see from this picture. However, not getting result in the sample might just be a chance. A few steps of optimization will solve that problem.

Look for the following steps:

1. Check for the sample concentration you are using and try to match it with other studies -- how large or how small quantity they have used in their case.

2. Check for the primer concentration because too large quantity of primer of sample might negatively influence the reaction.

3. Since your amplification curve looks little bit lower, and it doesn't appear to be that sharp to me, I would advise you to look for the annealing temperature and try to lower it a bit (something between 5 to 2 degree centigrade).

Good luck with that.