Feb 05, 2020
qPCR assay for detecting Batrachochytrium dendrobatidis
- Omneya Osman1,
- Johan Andersson2,
- Alexander Eiler1,
- Mats Töpel1,
- Tomas Larsson1,
- Tomas Larsson1
- 1eDNA solutions;
- 2Water Circle
Protocol Citation: Omneya Osman, Johan Andersson, Alexander Eiler, Mats Töpel, Tomas Larsson, Tomas Larsson 2020. qPCR assay for detecting Batrachochytrium dendrobatidis. protocols.io https://dx.doi.org/10.17504/protocols.io.baiticen
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: December 16, 2019
Last Modified: February 05, 2020
Protocol Integer ID: 31027
Keywords: Batrachochytrium dendrobatidis, qPCR
Abstract
The fungus Batrachochytrium dendrobatidis (Bd) was first detected in Norway in 2017, and thus indicate the arrival of an invasive black-listed species in the country.
Here we report the details of real time PCR assay which was used to screen for B. dendrobatidis from water samples collected from different locations in Norway.
Guidelines
Laboratory work space and equipment were sterilized by UV-light and DNase solution and 70% ethanol. Filter pipet tips were used in all steps of the laboratory work.
Negative controls of DNase/RNase free water were used in each qPCR assay.
Materials
MATERIALS
UltraPure™ DEPC-treated WaterThermo FisherCatalog #10813012
SsoAdvanced Universal Probes SupermixBio-Rad LaboratoriesCatalog #172-5280
Batrachochytrium dendrobatidis 5.8S ribosomal RNA (5.8S) genesig Standard Kit
Safety warnings
Handling high concentration of positive controls was performed in a post-PCR room which is physically separated from the pre-PCR room to avoid contamination.
Always add your samples first and seal them before adding the serial dilutions of positive control (standard) at the end.
DNA extraction
DNA extraction
DNA extraction was performed using Qiagen DNeasy power water sterivex kit. The quality of the extracted DNA was estimated using Nanodrop.
Qiagen DNeasy power water sterivex kit: https://www.qiagen.com/se/resources/resourcedetail?id=c5fe7d5f-070a-4ebe-ac04-4bbf05a13e91&lang=en
Internal DNA extraction control
When performing DNA extraction, it is often advantageous to have an exogenous source of DNA template that is spiked into the lysis buffer. This control DNA is then co-purified with the sample DNA and can be detected as a positive control for the extraction process. Successful co-purification and qPCR for the control DNA also indicates that PCR inhibitors are not present at a high concentration.
Internal control
4 µL of the internal control were mixed with lysis buffer as given in the extraction protocol.
Purification and amplification of the internal control DNA was tested as well as the presence of PCR inhibitors.
The primers are present at PCR limiting concentrations which allows multiplexing with the target sequence primers.
Amplification of the control DNA does not interfere with the detection of the B. dendrobatidis target DNA even when present at low copy number.
Real Time PCR
A B.dendrobatidis specific primer and probe mix is provided by Primerdesign Ltd which was detected through the FAM channel. The primer and probe mix provided exploits the so-called TaqMan® principle.
During PCR amplification, forward and reverse primers hybridize to the B.dendrobatidis DNA. A fluorogenic probe is included in the same reaction mixture which consists of a DNA probe labeled with a 5`-dye and a 3`-quencher. During PCR amplification, the probe is cleaved and the reporter dye and quencher are separated. The resulting increase in fluorescence can be detected on a range of qPCR platforms.
| Component | Resuspend in water | |
| B.dendrobatidis primer/probe mix (BROWN) | 165 μl | |
| Internal extraction control primer/probe mix (BROWN) | 165 μl |
Positive and internal controls
Safety information
* This component contains high copy number template and is a VERY significant contamination risk.
It must be opened and handled in a separate laboratory environment, away from the other components.
Resuspend the internal control template and positive control template in the template preparation buffer supplied, according to the table below:
| Component (heat-sealed foil) | Resuspend in template preparation buffer | |
| Internal extraction control DNA (BLUE) | 600 μl | |
| B.dendrobatidis Positive Control Template (RED) * | 500 μl |
To ensure complete resuspension, vortex each tube thoroughly.
Preparation of standard curve dilution series
Preparation of standard curve dilution series
1) Pipette 90 μl of template preparation buffer into 8 tubes and label them.
2) Pipette 10 μl of Positive Control Template (RED) into tube 2
3) Vortex thoroughly
4) Change pipette tip and pipette 10 μl from tube 2 into tube 3
5) Vortex thoroughly
Repeat steps 4 and 5 to complete the dilution series
| Positive control | Copy number | |
| Tube 1 Positive control (RED) | 1e6 per 5 μl | |
| Tube 2 | 1e5 per 5 μl | |
| Tube 3 | 1e4 per 5μl | |
| Tube 4 | 1 e3 per 5 μl | |
| Tube 5 | 1e2 per 5 μl | |
| Tube 6 | 1e1 per 5 μl | |
| Tube 6.5 | 5 per 5 μl | |
| Tube 7 | 1e-1per 5 μl | |
| Tube 8 | 1e-2 per 5 μl |
A standard with very low copy number (theoretically 0.1 cells per PCR reaction) was prepared to determine limit of detection of this assay.
PCR program for BD detection
PCR program for BD detection
| Component | μl | μl | |
| SsoAdvanced Universal Probes Supermix | 10 | 5 | |
| B.dendrobatidis primer/probe mix (BROWN) | 1 | 0,5 | |
| RNase/DNase free water (WHITE) | 4 | 1,5 | |
| Final Volume | 15 | 7 |
Pipette 5 μl DNA template if the total PCR mixture is 20 μl or 3 μl of DNA template if the total PCR mixture is 10 μl.
For negative control wells, we used 5 or 3 μl of RNase/DNase free water.
PCR program
PCR program
02:30:00
| Step | Time | Temp (C) | ||
| Enzyme activation | 2 min | 95 | ||
| Cycling x50 | Denaturation | 10 s | 95 | |
| DATA COLLECTION * | 1min | 60 |
* Fluorogenic data should be collected during this step through the FAM channel.
PCR for internal control
PCR for internal control
| Component Volume | Volume (μl) | |
| SsoAdvanced Universal Probes Supermix | 10 | |
| Internal extraction control primer/probe mix (BROWN) | 1 | |
| RNase/DNase free water (WHITE) | 3 | |
| Final Volume | 15 |
Same above PCR program was used but data is collected through VIC channel.
Pipette 5 μl of DNA template to reach 20 μl final volume in each well.
For negative control wells use 5 of RNase/DNase free water.
QPCR was performed in BioRad qPCR machine CFX96.
Analysis of the results was done by CFX maestro software
Interpretation of results
Interpretation of results
Expected result
Quantitation cycle (Cq)
Positive control template, 1e6 per 5 μl, is expected to amplify between Cq 16 and 23. Failure to satisfy this quality control criterion is a strong indication that the experiment has been compromised.
Where the test sample is positive and the negative control is positive with a Cq > 35, the sample must be reinterpreted based on the relative signal strength of the two results:
Internal PCR control
The Cq value obtained with the internal control will vary significantly depending on the extraction efficiency, the quantity of DNA added to the PCR reaction and the individual machine settings. Cq values of 28±3 are within the normal range.
When amplifying a B. dendrobatidis sample with a high genome copy number, the internal extraction control may not produce an amplification plot. This does not invalidate the test and should be interpreted as a positive experimental result.
| Target (FAM) | Internal control (VIC) | Positive control | Negative control | Interpretation | |
| ≤ 30 | +/ - | + | - | POSITIVE QUANTITATIVE RESULT calculate copy number | |
| > 30 | + | + | - | POSITIVE QUANTITATIVE RESULT calculate copy number | |
| > 30 | - | + | - | POSITIVE QUALITATIVE RESULT do not report copy number as this may be due to poor sample extraction | |
| - | + | + | - | NEGATIVE RESULT | |
| + /- | +/ - | + | ≤ 35 | EXPERIMENT FAILED due to test contamination | |
| + /- | +/ - | + | > 35 | * | |
| - | - | + | - | SAMPLE PREPARATION FAILED | |
| + /- | +/ - | - | + /- | EXPERIMENT FAILED |