Hi all,
How would you use qPCR to quantify the amount of a particular bacterial species in a sample experimentally?
I understand you could use specific primers for the 16s V region of a bacterial species e.g. Lactobacillus Reuteri but how would you then quantify the CT values generated from your sample?
For instance, would you include a standard curve of Lactobacillus reuteri on the plate? If so, how is this possible if you cannot culture the bacterial species?
Instead of culturing, is it possible to order in bacterial samples to use on a qPCR plate for quantification? e.g. Bacterioides?
You can PCR amplify and clone the 16S region of your bacteria of interest. Use the purified & digested plasmid to create a standard curve. If you calculate the exact copy number of your plasmid in the standard curve, then you can use it to determine exact copy numbers of your bacteria in your environmental sample (e.g. # bacteria genomes per 1 g of soil).
You can PCR amplify and clone the 16S region of your bacteria of interest. Use the purified & digested plasmid to create a standard curve. If you calculate the exact copy number of your plasmid in the standard curve, then you can use it to determine exact copy numbers of your bacteria in your environmental sample (e.g. # bacteria genomes per 1 g of soil).
I have a solution to this. It won't require any cloning. If you want to differentiate between different known bacterial species, then design the primers from conserved regions of the genome (obviously the amplicons have to be different for each bacterial species) where you know the sequence of the amplicon at least the exact length of the amplicon. I guess I should consider the fact that you know the amplicon sequence if you are doing Taqman assays (probe-based assays). Order the amplicon as oligonucleotide from any vendor (Thermo Fisher, IDT etc). Make a standard curve using the ordered know concentration of the oligonucleotide and then compare the experimental data with the standard one.
Thank you all very much for your helpful answers. Katie and Fahmid, please could you provide me with a protocol or a paper which outlines the methods you have suggested?
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