Dear prof.Jackwood:
Hello , i hop that you are fine ,i have a question regarding PCR-sample preparation and i hope if you could help :
Q) Why: Usually organs that rich in enzymes like ''liver & pancreas '' are not good for the Real time PCR /Rt-PCR ,because they may interfere with the PCR reaction especially if it is taken to diagnose an RNA virus like Avian influenza ...would you explain how they will interfere with the reaction ? and in which step/s exactly they may interfere ?

Regards

Ismail
* Last updated by: ismail on 3/23/2010 @ 4:40 PM *

Ismail - You don't need to post the same question in different locations. Once in the appropriate forum should be sufficient.

Hi Ismail,

There are dozens of PCR inhibitors, and I guess that we do not know all of them. Some have been well characterized but some others remain unknown in their mechanism of action. However, the modern extraction and purification reagents (commercial kits) are designed to avoid their interference. In my opinion the best approach to detect the presence of inhibitors at the sample level in a PCR run, is to include an internal positive control (IPC) in the PCR mix, that MUST be amplified even if the target sample is negative. If the IPC does not become amplified then you have no PCR reaction, and results are not valid. Please have a look to a short technical note attached (I found it in Internet)

I agree with Jamie. Attached is a paper where we prepared an internal control for our IBDV RT-PCR assay. It works very well but you have to also remember that it competes with the viral genome so the internal control can sometimes lower the sensitivity of the assay. This is only a problem when the quantity of virus in a sample is very low.

Thanks Daral. Nice paper. It is also advisable to read the OIE manual dealing with validation of molecular assays used for veterinary diagnostics.

http://www.oie.int/fr/normes/fmanual/A_00012.htm