Sep 18, 2024
Quantification of fungal ITS Gene Copies Using ddPCR (EvaGreen-based assay: ITS1f-ITS2)
- 1Soil and Water Research Infrastructure
- Anaerobic and Molecular Microbiology Lab, Biology Centre CASTech. support email: eva.petrova@bc.cas.cz
Protocol Citation: Eva Petrova, Roey Angel 2024. Quantification of fungal ITS Gene Copies Using ddPCR (EvaGreen-based assay: ITS1f-ITS2). protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmx13ol3p/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 29, 2020
Last Modified: September 18, 2024
Protocol Integer ID: 37584
Keywords: Absolute quantification, ddPCR, 16S rRNA
Abstract
The protocol was designed to quantify microbial eukaryotic fungi using ITS region copy number evaluation by Droplet Digital PCR technology (ddPCR) from Bio-Rad company.
For the assay, we are using a universal fungal ITS rRNA primer pair:
ITS1f 5'- CTT GGT CAT TTA GAG GAA GTA A -3', 38 bp upstream of ITS1 from White et al., 1990
ITS2 5'- GCT GCG TTC TTC ATC GAT GC -3', identical to ITS2 from White et al., 1990until now because of its extreme length variability among the fungi species. In fact, this variability introduced a bias to final copy numbers estimated by qPCR,.
The same ITS1f and ITS2 primers are recommended for use for fungi identification by the Earth Microbiome Project.
Note
Internal Transcribed Spacer (ITS) has not been possible to use for fungi quantification until now because of its extreme length variability among the fungi species. In fact, this variability introduced a bias to final copy numbers estimated by qPCR, which was overcome here with the ddPCR method.
Attachments
Guidelines
- The crucial steps that can influence the final assay results a lot are precise pipetting, mixing and dilutions!
- Keep in mind that ddPCR technigue do not posses as wide dynamic concentration range as qPCR does. You can easily "overload" the reaction with too much template DNA putting in. In that case you will see only positive droplets at the end and no negative and system will not be able to calculate copy number for them. As a consequence, you usually have to dilute your template DNA more than for qPCR experiments. Ideally fit inside the range of 101 - 104 coppies of target gene. That is why we usually test several dilutions of few samples in advance to see "where we are".
- One have to also keep in mind that using ddPCR technology you must work within the format of eight. If you do not fulfil all the wells in a cartridge you still have to put the reagencies inside the empty wells as well. So, think economically before you start.
Materials
MATERIALS
PCR Plate Heat Seal foil piercableBio-rad LaboratoriesCatalog #1814040
ddPCR 96-well platesBio-rad LaboratoriesCatalog #12001925
QX200™ ddPCR™ EvaGreen SupermixBio-rad LaboratoriesCatalog #1864033
Automated Droplet Generation Oil for EvaGreenBio-rad LaboratoriesCatalog #1864112
Protocol materials
PCR Plate Heat Seal foil piercableBio-Rad LaboratoriesCatalog #1814040
Step 9
PCR Plate Heat Seal foil piercableBio-Rad LaboratoriesCatalog #1814040
Materials
ddPCR 96-well platesBio-Rad LaboratoriesCatalog #12001925
Materials
QX200™ ddPCR™ EvaGreen SupermixBio-Rad LaboratoriesCatalog #1864033
Materials, Step 2
Automated Droplet Generation Oil for EvaGreenBio-Rad LaboratoriesCatalog #1864112
Materials, Step 7
ddPCR 96-well platesBio-Rad LaboratoriesCatalog #12001925
Step 2
PCR Plate Heat Seal foil piercableBio-Rad LaboratoriesCatalog #1814040
Step 4
Safety warnings
Protect probe from light.
Before start
Take all the reagencies out of a freezer and let them temperate to room temperature.
ddPCR reaction mixture
ddPCR reaction mixture
20m
20m
All reagencies must be equilibrated to RT (do not keep them on ice). Mix each of them properly before use.
20m
| Reagent | Final conc. | 1 tube (22 μl) | plate (22 μl x 100) | |
| PCR H2O | 8.6 | 860 | ||
| QX200 ddPCR EvaGreen Supermix | 1x | 11 | 1100 | |
| ITS1f (10 μM) | 0.1 μM | 0.2 | 20 | |
| ITS2 (10 μM) | 0.1 μM | 0.2 | 20 | |
| Template | 2 | 2 x 100 |
Prepare the master mix according to the number of samples and mix several seconds by vortexing. Transfer mix into 96-well plate à 20 µl.
20 µL master mix per well
ddPCR 96-well platesBio-rad LaboratoriesCatalog #12001925
QX200™ ddPCR™ EvaGreen SupermixBio-rad LaboratoriesCatalog #1864033
Note
Tip: use a mechanical or electronic dispenser (for ex. Multipette, Pipettman, or multichanel pipette) during this step to speed up the work.
Add 2 µl of your DNA sample into each well
2 µL of examined DNA per well
Seal the plate (180°C, 5s) with pierceable aluminium foil.
PCR Plate Heat Seal foil piercableBio-rad LaboratoriesCatalog #1814040
00:00:05 sealing at 180°C
Let the foil cool down and mix the plate vigorously by vortexing 30 s - 1 min.
00:00:30 vortexing
Droplets generation by AutoDG
Droplets generation by AutoDG
Put all the staff (cartridges, tips, sealed plate with samples and empty 96-well plate in cooling stand) in corresponding amount inside the AutoDG machine (Bio-Rad).
Note
Per one sample there is a need of two pipette tips!
This is an example how it should look like inside the AutoDG before strating droplets generation.
Make sure there is a right oil bottle (Automated Droplet Generation oil for Probes) connected to the system.
Automated Droplet Generation Oil for EvaGreenBio-rad LaboratoriesCatalog #1864112
Choose a position of samples on touch screen and start dropplets generation. Wait after its finished.
Note
System will you announce automatically about success or failure of droplets creation after procedure is finished. Nevertheless, every time make also a visual inspection of droplets. Two separated phases should be visible. Upper part with dropplets and lower clear oil phase.
selection of a columns within the plate where the samples are positioned
This is how it should look like inside the AutoDG after droplets generation.
plate after droplets generation - two phases visible in each well with sample
Take the plate with droplets out of the machine and seal it with pierceable aluminium foil (170°C, 3s).
PCR Plate Heat Seal foil piercableBio-rad LaboratoriesCatalog #1814040
00:00:03 sealing at 170°C
Put immediately the sealed plate into PCR cycler.
Note
Droplets are unstable at this stage. Proceed to next step as fast as possible. After PCR droplets become stable and can be kept at fridge for some time (one day) before measurement.
Clean AutoDG machine, waste used consumables.
PCR program
PCR program
3h
3h
1. 95◦C –5′
2. x 40 {
a. 95◦C – 30′′
b. 52◦C – 2′
}
3. 4◦C – 5′
4. 90◦C –5′
5. 10◦C –hold
Set ramp rate for each step to 2°C/sec!
Set reaction volume to 40ul.
40 µL reaction volume
Note
- After run is finished check if there are still two phases present
- Let the plate cool down (droplets will not be so sticky and will be more easy to analyse).
Droplets reading
Droplets reading
Note
Switch on the reader 30 min before measurement.
Droplet reader with a plate after PCR already placed inside the metal holder
Set up the QuntaSoft experiment as follows:
Exp. type: Absolute quantification (ABS)
Supermix: QX200 ddPCR EvaGreen Supermix
Target1: Ch1 (for FAM labeled probes)
Define the position of ech sample.
Check the levels of reader Oil and waste - green control (bottles are physically accessible from machines left side).
Note
If the instrument was not in use longer than week it has to be Primed first (oil flushed).
Start measurement.
Note
After it finishes go over the results and see how many droplets were executed for each sample. To get reliable results total droplet count should be above 12.000. We usually have got 18.000 - 20.000 droplets analyzed per sample.
an example of total droplets count (positive together with negative ones) being generated and read
Analysis
Analysis
Set up the treshold just above negative control sample in order to distinguish positive (containing PCR products) from negative (do not contain any PCR product) droplets.
Note
Quanta software will automaticaly calculate copy number of target gene for each sample by Poisson distribution algorithm. For that calculations at least some of the negative droplets are necessary. If the sample contains only positive population it can not be evaluated.
An example of ITS Fungi copy numbers data analysis. Last two samples on the left graph are negative (NTC) samples by which a treshold was set up.
Export .csv file with concentrations (copies/ul) that are posessing you the number of copies in 1ul of reaction. To obtain number of copies in 1ul of your input DNA you have to recalculate:
no. of copies in 1ul of input DNA = (concentration value x 22)/ volume of DNA