We have been using a set of primers for sometime now for HCV RNA detection using conventional PCR and qPCR. All was fine; Cts for HCV positive samples, no Cts for negative samples and when water was ran as a mock sample and the same for conventional PCR in terms of bands on gel. However, all of a sudden we started getting Cts with negative samples and water with q PCR and smears with conventional PCR. Attached is an example of one of these runs (Primer RG-1)

It is worth note that we started experiencing this, while using the same stock of primers, probes and the same mastermix. So its not a change of lot or source.

We are almost 100% sure its not contamination, particularity since a positive band is not obtained, and because we opened new stocks from all our components and still found the same.

We also tried using another set of primers (that we have been using in the past for HCV detection) still got smears but that looked quiet different. (Attachment primer RG-2).

Are the primers the culprit? could it be that all of a sudden we need to revise our PCR conditions, thermal profile?....

Thanks