detection of non-specific bacteria at higher CT value

[goh]

hi, i've found a problem in qPCR detection. my qPCR assay was able to detect non-specific bacteria although at higher CT value (e.g. 30). I've carried out the BLAST search on the specificity of the primer and the result shows that no mismatch for the primer. may i know how to overcome this problem? thanks

[Aga]

Before I can try to help you I need to know whether you're doing probe based assay or SYBR Green and how do you know it's non-specific bacteria, or even bacteria at all, that you detect at higher Ct values?

[goh]

Thanks Aga. For your information, I'm using the probe based assay. For specificity test, I've tested on the purchased pure culture or requested pure culture from the authors. For example, my target is enterococcus bacteria. I've tested my qPCR assay on enterococcus pure culture bacteria as well as other pure culture bacteria such as E.coli, Streptococcus, Lactococcus, etc. Hope you can help me. Thanks in advance.

[Aga]

The problem is such that even if you Blast your sequences and think your primers can't bind to any other but specific sequences there is always a chance you may have some unspecific products. It is because not all sequences are known and available in GenBank.

The above would occur if there is some kind of contamination - the source can be everywhere. You will have to be sure everything is sterile including pipettes, working area, reagents etc. It is desirable to have completely separate working area for qPCR.
Use filter pipette tips - often pipettes suck up some liquid and if they are not sterilised they cause problem.

Sadly, you can also be the source of contamination.

Other reasons than contamination:
Have you performed the same assay using SYBR Green I?
I usually do that before I set the protocol for probes. This is because SYBR Green will tell you whether there are any non-specific products in the background or primer-dimers.

The chance is usually very small, but you need to be sure that your probe does not bind to any of the background products or primer-dimers. This would cause little increase in fluorescence usually at higher CTs.

These are my ideas. I had similar problem as you before with TaqMan probes. I concluded it was contamination that was the cause and I eliminated all possible sources of contamination plus forced my supervisor to change HEPA. And it worked well.

[goh]

Thanks Aga. I will try to test my samples using SYBR Green I. I guess my non-specific detection may caused by the contaminations. I will take your suggestions in my future experiments. Thanks for your suggestions. Hope can report good news to you soon. Thanks.