It can be done, but the end result is not going to be very relevant.

First, growing your sample in a medium will already limit the species that you will find after culturing, and the abundance ratios will probably already be very different from those in stool. Certain bacteria will grow faster than others, depending on the type of medium you choose, so the ratios of those bacteria will be higher than in the original sample.

Second, if you just do a PCR on grown bacteria, without DNA purification (but maybe just a simple boiling step), you will only detect the bacteria that are very easy to lyse. The first PCR step is a near-boiling step, so you will break open some bacterial species, but many others are harder to lyse and won't be detected.

So you could do a qPCR and get results, but the results will be very different from the abundances in the original sample. Even if you do a qPCR on a gene that all bacteria have, such as the 16S rRNA gene, the number of bacteria (or rather 16S copies) is not going to match that of the original sample. I am not sure how that would be relevant, but maybe it makes sense in your specific application.

Anyway, I would highly recommend skipping the culturing step and doing a DNA purification directly on the stool sample. The results from a qPCR on that DNA would be much more relevant to the real abundances in the original sample.

I hope this helps!

First, I don´t know if you already identify your strain, you can select differential mediums. Then from a colony take a sample, annd amplify it

Could you clarify what you mean by optimizing your reaction? Are you not getting the CT's you'd expect? Is your curve working well? Are you performing each reaction in triplicate?

Just a couple things...I would want a little more understanding of cell number. Count your cells at your 10x dilution to get an idea on how many bacterial cells are required not only for (+) amplification, but also for (-) amplification. Knowing the number of cells will allow you to determine with greater accuracy expression levels on a per cell basis (provided that's information you'd like to know). Also, if you aren't already doing it, I would wash your cells a couple times in sterile water or PBS to remove any traces of media. Third, believe it or not but simply experimenting with different qPCR kits can have drastic results on your outcome. If you are able and have the kits available, you might try that. What are you using to generate your standard curve?

Good Luck

Barry

It can be done, but the end result is not going to be very relevant.

First, growing your sample in a medium will already limit the species that you will find after culturing, and the abundance ratios will probably already be very different from those in stool. Certain bacteria will grow faster than others, depending on the type of medium you choose, so the ratios of those bacteria will be higher than in the original sample.

Second, if you just do a PCR on grown bacteria, without DNA purification (but maybe just a simple boiling step), you will only detect the bacteria that are very easy to lyse. The first PCR step is a near-boiling step, so you will break open some bacterial species, but many others are harder to lyse and won't be detected.

So you could do a qPCR and get results, but the results will be very different from the abundances in the original sample. Even if you do a qPCR on a gene that all bacteria have, such as the 16S rRNA gene, the number of bacteria (or rather 16S copies) is not going to match that of the original sample. I am not sure how that would be relevant, but maybe it makes sense in your specific application.

Anyway, I would highly recommend skipping the culturing step and doing a DNA purification directly on the stool sample. The results from a qPCR on that DNA would be much more relevant to the real abundances in the original sample.

I hope this helps!