Q-PCR for mycoplasmas - performance is way to low (Jan/17/2008 )

Hi,

Im setting up a Q-PCR to quantify mycoplasma's on genomic DNA. Im using primers that have been tested & tried for various species & recognise these with similar efficiency.
Theoretically, I should be able to detect 1 cell / reaction, realistically perhaps the limit of detection would be 10 cells. So far im not able to get below 100 cells / reaction, with a Ct of about 32. For some species Im even having trouble detecting 10.000 copies reliably (although my primers are picking them up after culturing without any problem).

Im isolating the DNA with a Qiagen-blood kit (which I think might be the problem) and have already switched to eluting in water instead of TE (EDTA might be messing up the reaction),
using 5 ul DNA input (representing a range of mycoplasma genomes from 10^5 to 10^0) / 50 ul reaction volume,
running the reaction on a ABI 7300 in the ABI Sybrgreen mix,
with 15pmol of each primer
on a standard program.

Has anybody tried Q-PCR on genomic DNA from mycoplasmas? (or does anyone have tips for quantifying genome copies by Q-PCR in general)
Can anyone recommend a DNA extraction method for genomic DNA which works well for Q-PCR applications?
of course any other tips & pointers are very welcome!

Thanks in advance,
Stape

Mycoplasmas have no cell wall, and are easily lysed by simple heating. I would avoid attempts to "purify" the DNA at all, and simply try cells directly in the PCR reaction, extending the initial denaturing phase by a few minutes. You might also try lowering the extension (not annealing) temperature to 66, since the very high AT content of mycoplasmas can make extension through very high AT rich regions fail at 72, or even 68.

Thanks alot, I'll give it a try next week & post the results here!!