I know this is a very broad question, but in your experience how sensitive is qPCR?
It is reported that qPCR can detect even 1000 CFU/ml or less of bacterial species in human samples, and brochures of qPCR manufacturers show that it is quite sensitive.
But, generally speaking (regardless of the type of master mix you are using), have you ever had sensitivity problems with qPCR and what were the lower limits of detection for you?
Hi Sammy
There is no doubt about qPCR sensitivity of detection. The point is this method has its own weakness. It is expensive (in absolute approach!) and will count dead and alive cells. CFU is more realistic but in case of a hard-to-culture species would be a useless method. My recommendation is to choose CFU over qPCR for counting if you can culture easily.
Good luck
Hello, depends what accuracy you looking for. The classical microbiological decimal dilution method will provide more accuracy than Q-PCR for CFU/ml detection.
See info attached may be useful for you.
Good luck!
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3517291/
Hi,
Both method are good options. Depend what do you want.
With PCR you can detect 10 or 100 bacterias in 0,1 g of tissue. Success will depend on the quality of the sample (fresh biological material) and experience of the analyst.
I suggest using UFC and PCR in your work for the starting.
Hi Sammy,
Depending on your target bacterial gene and the specific tissue type you're processing, it could be straightforward or challenging. If you're using 16S as the target, you could certainly pick up enough copies to translate to 1000 CFU/mL. If your standard curve can consistently produce Ct values for 10^3 and 10^2 gene copies and not have to exclude them, then you could certainly pick it up. Maintaining the lower gene copy standards has been the most challenging for myself, since ensuring consistent dilution to these lower concentrations is difficult. I've targeted 16S and other single copy bacterial genes and have detected down to the 10^2 level, from environmental samples (soil and water) ... when the standards work.
Purification of the isolated nucleic acids may also be necessary, to ensure that your starting concentrations are high enough. If necessary, Norgen Biotek has a DNA and RNA clean-up and concentration kits available that will improve your chances of detection, if you're not expecting high bacterial concentrations from the human tissue samples.
Cheers,
JP
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