If double strand DNA is generated during qPCR, fluorescence intensity increase by SYBR green. Thus, Ct value is affect by non-specific binding of primers; primer dimer, non specific PCR amplicon.

i recommand multing curve analysis or taqman probe.

Dear all, thank you for your comments and suggestions. In my particular case I can only use 16S, since the genome (and species) of one of the strains is still unknown.

Also, I checked for unspecific amplification with negative controls and this doesn't seem to be my case.

Your insights have been very helpful! I guess contribution from dead cells and non optimal growth conditions could be the case.

Hi Ana,

CFU is usually not really suited for cell counting, because it often underestimates the original cell number. As it's name suggests, it gives you a direct count of colony forming units, not the number of individual cells. One colony is not necessarily formed from a single bacterial cell, but from many cells clustered together.

Actually, there are many species that barely occur in a single cell form, they form clusters with the morphology of e.g. sarcina, diplococci, staphylococci etc. Colonies of these are usually formed from these clusters, and not from a single cell. I don't know your strains, but this could explain the difference between your qPCR and estimates from CFU.

hi,

All comments above are useful and they could be a possible reasons. Just i wanted to add  more point about how to quantify  the live cell only by qPCR  using different dyes instead of sybr green  like   Ethidium Monoazide and Propidium Monoazide. have a look to this paper.

Comparative Analysis and Limitations of Ethidium Monoazide and Propidium Monoazide Treatments for the Differentiation of Viable and Nonviable Campylobacter Cells

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3993131/#

Good luck

Hello Ana,

I would add to the above comments that you should do a a lit search on the specificity and the quality of binding of both your fluorescent tags with your bacteria as swell as your primers with your specific strains (just because they work may not mean they work really well).

PCR bias is not discussed nearly enough in the world of molecular biology (and microbiology), I would check this paper out:

Ruijter, J. M., et al. "Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data." Nucleic acids research 37.6 (2009): e45-e45.

Also depending on the type of bacteria you are working with it is also a chance for Polyploidy.  If you find a consistent ratio of over estimation by qPCR then it could help  lead you to your answer.

Soppa, Jörg. "Polyploidy and community structure." Nature microbiology 2 (2017): 16261.

Cheers,

Ceth

Hi Ceth,

Thank you very much for your ideas and for the papers. I will look into that.

Cheers,

Ana

How do you determine your cfu count? If you are doing 10-fold serial dilutions, perhaps a simple arithmetical error explains your 10x (1 log) more copies than CFU.