Mar 02, 2020
The detection of freshwater pearl mussel
- Omneya Osman1,
- Alexander Eiler1,
- Mats Töpel1,
- Tomas larsson1
- 1eDNA solutions
- eDNAsolutionsTech. support phone: +0700264843 email: omneya@ednasolutions.se
Protocol Citation: Omneya Osman, Alexander Eiler, Mats Töpel, Tomas larsson 2020. The detection of freshwater pearl mussel . protocols.io https://dx.doi.org/10.17504/protocols.io.974h9qw
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 06, 2019
Last Modified: March 02, 2020
Protocol Integer ID: 30684
Keywords: freshwater pearl mussel, qPCR
Abstract
qPCR assay to detect freshwater pearl mussels Margaritifera margaritifera in order to monitor populations in their natural environment.
Attachments
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 #10813999
Qiagen DNeasy power water sterivex kitCatalog #14600-50-NF
STEP MATERIALS
SsoAdvanced Universal SYBR® Green SupermixBioRad SciencesCatalog #172-5270
SsoAdvanced Universal SYBR® Green SupermixBioRad SciencesCatalog #172-5270
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 was performed using Qiagen DNeasy power water sterivex kit. The quality of the extracted DNA was estimated using Nanodrop.
Qiagen DNeasy power water sterivex protocol can be found in this link: https://www.qiagen.com/se/resources/resourcedetail?id=c5fe7d5f-070a-4ebe-ac04-4bbf05a13e91&lang=en
Primers
Primers
Stoeckle et al., 2016 designed primers targeting 16S rRNA gene of Margartifera margaritifera.
The sequence of the primer set is:
MarMa_16S2.1: 5’- GCAACACGGAAAACCCC TG -3’
MarMa_16S1.2: 5’- GGCT GCGCTCATGTGAATTA -3’.
The concentration of freshwater pearl mussel standard was measured by PicoGreen assay (http://tools.thermofisher.com/content/sfs/manuals/PicoGreen-dsDNA-protocol.pdf) before preparing standard serial dilutions.
Serial dilutions, 10, 1, 0.1, 0.01 and 0.001 ng, were prepared to generate standard curve in each qPCR run.
Unio pictorum or Äkta målarmussla was used to test the specificity of qPCR detection.
Plate setup
Plate setup
Setup and design your qPCR plate in the template of qPCR machine before preparing your qPCR run.
At least 3 or 4 replica of standard dilutions should be included in each run.
Three replica of each sample was performed.
PCR reaction
PCR reaction
A master mix was prepared and calculated according to the number of standard dilutions, samples and negative controls to run.
The PCR mixture per sample was as follows:
| Reagent | working sol | final conc | µl | |
| SsoAdvanced Universal SYBR® Green Supermix | 2x | 1x | 10 | |
| MarMa_16SF primer | 10 uM | 0,25 uM | 0,5 | |
| MarMa_16SR primer | 10 uM | 0,25 uM | 0,5 | |
| DEPC-water | 6 | |||
| DNA template | 3 | |||
| ∑ | 20 | |||
Sample or qPCR plate should be carefully sealed with no marking on the tube to facilitate plate reader step.
PCR program
PCR program
| Amplification step | Time | Temp | ||
| Enzyme activation | 15 sec | 98 °C | ||
| Denaturation | 10 sec | 98 °C | 40 cycles | |
| Annealing | 10 sec | 60 °C | ||
| Extension | 20 sec | 72 °C | ||
| Melt curve analysis | 63 to 99°C |
QPCR was performed in BioRad qPCR machine CFX96.
Analysis of the results was done by CFX maestro software
QPCR efficiency, limit of detection and quantification range of standard were checked for each run.
Results can be evaluated through data analysis windows where there is a quantification, standard melt curve and RFU spreadsheet.