Hi,
I'm using qPCR as a mean of detecting air sampled bacteria, but the thing is that in my bacterial stock I have much more genome copies than CFU, like in 1mL I have 1x10^6 CFU and in the qPCR I detect 1x10^9. So I began to wonder if I'm calculating the genome copies in a wrong way. Here is how I do it: I extract bacterial DNA and then quantify it with nanodrop, then I do a serial dilution of this and use it as a standard curve for qPCR. Based on the curve generated, I then calculate the DNA mass of my samples with the CT I got. Based on this DNA mass I calculate the genome copies using this equation: =(DNAmass*6,022E+23)/(GenomeSize*1e9*660).
Any thoughts?
What is your target gene?
You have to consider that some genes like 16s have more than one copy number in a bacterial cell.
Check your gene for its copy number.
In other hand consider there is bacteria can not be cultured, maybe these types of bacteria have your gene.
So there is nothing odd with your result.
Good luck
Hi Alireza Mordadi,
I should have mentioned that, I'm working with M. bovis BCG and targeting an insertion gene (IS6110) that only has one copy in the genome.
I also though about having bacteria that are viable but non culturable, since it is a mycobacteria it would not be that surprising, but the difference from 1 million CFU to 1 billion is way to big.
I also considered that there could be free DNA in my stock, but I do find hard to believe that this DNA would not be metabolize or degraded.
Are you sure about the copy number?
In my knowledge in general IS elements at least have tow sequence for i.e one in the start and on in the end of insertion sequence.
Alireza Mordadi , yes I am sure of the copy number, this is the strain used as a vaccine for tuberculosis, it has been fully sequenced.
When doing qPCR, you have to factor in the number of copies of your target gene per genome of bacteria. This is because the gene targeted by your primers may have more than one copy of such gene per genome. It becomes a problem when your genomic DNA is from environmental sample. Moreover, remember that PCR can amplify DNA originating from live or dead organism. Also there are many organisms that can not be cultured but PCR will pick them up which makes it difficult to compare results from culturing and qPCR.
Try to check copy number with digital PCR (BioRad, Constallation or RainDance-BioRad), it gave usually better data. Also direct calculation of bacteria number with special cell counters can help.
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