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improving sensitivity of one step TAQMAN REal time PCR - (Aug/15/2012 )

Hi there

I am trying to optimize a Taqman REaltime PCR assay with hepatitis delta viruses (RNA virus). My system generally works OK but I have to detect the lower copies of the Virus. My lower limit of detection is about 10000 copies (10^4) at the moment. I have optimized dNTP, enzyme, Mn+2 concentrations, and played a bit with the temperatures. Does anybody have an idea for a better detection limit of lets say 1000 (10^3) copies for instance?

Thanks in advance


More primers.
More efficient primers.
In one step, is there also an option to degrade RNA after reverse transcription? Sometimes the RNA can inhibit reaction.
Also in this respect, maybe try some additives, that may overcome such inhibition problems, HDV has higher GC content in some areas so maybe some secondary structure relaxing aditives like DMSO could also help.