Which fungal marker use to quantify fungal population via qPCR?

General Discussion
1

Hi guys
I want to take rhizosphere and soil samples and have a notion of how much fungi and bacteria there are in those samples.
For bacteria, I'm pretty sure that 16s will do the trick.
But for fungi, I'm a little in the dark. Should I use 18s or ITS will do what I want (quantify how much fungus there are in my samples) ?

2

Good afternoon,

To quantify fungi in your samples, use ITS. While 18S can be used for fungal identification, the ITS regions are more variable and provide better resolution for fungal community analysis and quantification.

(And when you get to the taxonomy classification step, consider using the pretrained Unite database I've made for ITS!)

3

Hi @moisessfeitosa ,

I have used fungal ITS primers quite a lot for QPCR. It is commonly used for this.

However, there are some things to keep in mind:

  1. many fungal primers also amplify other eukaryotes, so depending on your sample type and prep method you should keep this in mind. For rhizosphere I would expect a fair amount of plant DNA would be present, though if you are also doing ITS sequencing you should see approximately how much is plant. The fungi-specific ITS primers, on the other hand, tend to have reduced coverage of fungi as well, so could underestimate.
  2. The ITS domain is multi-copy and has variable copy number in fungi. So QPCR with ITS primers will quantify the number of copies, not the number of cells. A standard curve with a single strain will not really help, as you will then measure against the copy numbers per cell for that strain, which is likely not representative of all strains in your samples. A single-copy gene may be more appropriate (but I am unaware of any universal fungal QPCR primers for such a gene, so please let me know if you find one!)

It will! 16S is very commonly used for bacterial quantification via QPCR. However, the same caveats that apply to ITS also apply to 16S (re: cross-amplification of chloroplast and mitochondrial DNA and being a multi-copy gene).

I hope that helps!

3 Likes
4

Thank you, so much :slight_smile:

5

Thank you. Your answer has helped me a lot.