Hello everybody.
I have a sample from biofilm forming bacteria and I need to quantify (1) 16S rRNA gene copy numbers from each bacterial species within this sample and (2) antibiotica resistance genes, especially against Cloxacillin. Can please someone say, whether my thoughts for that matters are right?
1. RT-PCR with standard 16S rRNA primers to capture as many bacterial species as possible, followed by 16S rRNA sequencing and comparison of the obtained bacterial 16S rRNA gene sequences with databases.
2. Now we know the panel of target organisms, so we can amplify the PCR product with primers for hypervariable regions (to increase a selectivity) to obtain information on species composition. The housekeeping genes with a known concentration can be added as a referance into each sample. So you can calculate the 16S rRNA gene copy numbers. I also read that a treatment with a shrimp nuclease can increase a sensivity of this test? When I use the primers for the hypervariable regions, where can I find a database which primers exactly do I need?
3. Back to the antibiotica resistance genes. Does anybody know any papers or have any suggestions how to increase a sensitivity for the detection of antibiotica resistance genes within the bacterial community using qRT-PCR?
This sounds like about the best approach. However, don't expect to achieve accuracy better than +/- a factor of ten. As far as regards the primers, you'll probably have to design your own.
What are you trying to measure? rRNA gene copies per genome or number of cells of the various members of the community?
If you're after counting cells, I would use the rRNA sequences to identify the bacterial species and then use a housekeeping gene as target. It's easier to achieve specificity.
Lastly - so-called universal PCRs are biased. Don't rely on just one, or you may miss important species. If your biofilm community is very complex you may need to resort to high-throughput sequencing, but this will cost you about $10 000 and the bioinformatics is a bit daunting.
Here's a naive question : why do you want on RNA samples instead of DNA ? You'll also have informations about gene copies per genome if you analyse DNA.
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