Detection limit of a conventional PCR - (Mar/10/2011 )
Hi....
Would be most grateful if someone can let me know the acceptable range of detection limits for a conventional PCR targetting intracellular bacteria residing within circulating endothelial cells. If this is too specific, even the detection limit range for a conventional PCR is fine...
I also need to have a reference for this too
The detection limit (DL) of conventional PCR has as much to do with your detection method as it does the actual PCR itself. If you are using ethidium bromide the DL will be around 10 nanograms. If instead you use more "modern" stains like SYBR Green, you should be able to detect less than 1 nanogram, even 100 picograms (depending on how well you prepare your gel, and how thin it is).
On a more technical level, assuming you start with a single bacteria, and you are able to extract 100% of its DNA (which you never can), and assuming your PCR reaction is 100% efficient, which it never is, and you run 30 PCR cycles (which equals to a billion-fold amplification), then you will have the equivalent of 2 nanograms of PCR product. In other words, theoretically your DL should be around 10 bacteria.
This of course is only theoretical and it depends on a lot of factors. Unfortunately I do not have a reference for these calculations.
ivanbio on Fri Mar 11 01:02:10 2011 said:
The detection limit (DL) of conventional PCR has as much to do with your detection method as it does the actual PCR itself. If you are using ethidium bromide the DL will be around 10 nanograms. If instead you use more "modern" stains like SYBR Green, you should be able to detect less than 1 nanogram, even 100 picograms (depending on how well you prepare your gel, and how thin it is).
On a more technical level, assuming you start with a single bacteria, and you are able to extract 100% of its DNA (which you never can), and assuming your PCR reaction is 100% efficient, which it never is, and you run 30 PCR cycles (which equals to a billion-fold amplification), then you will have the equivalent of 2 nanograms of PCR product. In other words, theoretically your DL should be around 10 bacteria.
This of course is only theoretical and it depends on a lot of factors. Unfortunately I do not have a reference for these calculations.
Thanks a lot. Much appreciated. I do need a referance though, as I am discussing this for my thesis, will try to find one.