I understood that to generate a standard curve for a certain bacteria, the Ct values should be plotted relative to corresponding serial 10-fold dilutions of template DNA of known concentration extracted from representative cultures. But, how can I then convert the DNA concentration I'll get (ng/ul) into CFU/gram of stool?
Note: attached the two main references most related articles refer to.
Hello,
To convert the Ct of the qPCR in "CFU" you need to do another type of calibration curves, using 10 fold dilutions of your target bacteria followed by DNA extraction for each dilution. After that you will have a linear relation between the Ct and the number of bacteria. You have to keep in mind that this relation is not always true and depend of the inhibitor in the PCR, the state of the bacteria and lot of other parameter, but you have something approaching.
Alternatively you can calculate a relation between the number of copy (and not the quantity of DNA) in your qPCR reaction and the estimated number of target in your bacteria (for example one gyrB target by E. coli cell).
For the other part of the conversion (ng to g of stool), you just need to know the quantity of stool you have extracted and the relative part you use for your PCR.
Does it help you?
Hello,
To convert the Ct of the qPCR in "CFU" you need to do another type of calibration curves, using 10 fold dilutions of your target bacteria followed by DNA extraction for each dilution. After that you will have a linear relation between the Ct and the number of bacteria. You have to keep in mind that this relation is not always true and depend of the inhibitor in the PCR, the state of the bacteria and lot of other parameter, but you have something approaching.
Alternatively you can calculate a relation between the number of copy (and not the quantity of DNA) in your qPCR reaction and the estimated number of target in your bacteria (for example one gyrB target by E. coli cell).
For the other part of the conversion (ng to g of stool), you just need to know the quantity of stool you have extracted and the relative part you use for your PCR.
Does it help you?
I agree with Laurent,
You have to take in account that you are STIMATING the CFU, but it is not exactly the same.
If you now the ng you can convert en genome copy number, that is more or less similar to CFU (but not exactly the same). You can convert it here:
http://cels.uri.edu/gsc/cndna.html
You need to know the ng and the genome size. In Pubmed it should be available. The only thing you should consider is the gen copy number which is the target in your PCR. If there is only one copy, you have a direct estimation. If not, you should divide the result by the gene copy number of your target gene.
Finally, if you know the gr of stool you used for the DNA extraction you can converse to cfu/gr
I agree with Hector and Laurent. Exactly how you do the calculation depends on whether your template is total genomic DNA or a plasmid clone or other subgenomic fragment. If it is total genomic DNA, you convert the amount in ng to genomic copies (genomic units, GU) by dividing by the weight of the chromosome in ng (d0n't forget that it's double-stranded!). If it's plasmid, you divide by the weight of the plasmid. If your gene is single-copy (most bacterial genes are), the number of plasmid copies equals the number of chromosome copies, which in turn equals (to a first approximation) the number of cfu. If you are using a multi-copy gene, like 16s rRNA, you need to correct for the number of genomic copies: GU = total number of copies / copies per genome.
Note that it is more correct to report your results in GU than CFU. This is because qPCR makes no distinction between live and dead cells. In addition, in fast-growing cells, genes near the replication origin may be up to 8x more abundant than genes near the terminus, so there may be up to 8 GU per CFU.
Thank you very much Dr. Hector Dr. Laurent and Dr. Andrew for your clarification and explanations. I was really confused due the interchangeable use of the terms CFU and target genomes in many related articles.
you also read my publications if you really want to express your results in CFU
Article Use of Accuracy Profile Procedure to Validate a Real-Time PC...
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