Feb 03, 2025
Detection of marbled crayfish Procambarus fallax
- Alexander Eiler1,
- Eivind Stensrud1,
- Omneya Osman2
- 1eDNA solutions AB;
- 2IVL Swedish research Institute
- eDNAsolutionsTech. support phone: +0700264843 email: omneya@ednasolutions.se
Protocol Citation: Alexander Eiler, Eivind Stensrud, Omneya Osman 2025. Detection of marbled crayfish Procambarus fallax. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g78199lwz/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: December 28, 2020
Last Modified: February 03, 2025
Protocol Integer ID: 45879
Disclaimer
Use at own risk!
Abstract
Taqman QPCR assay for marbled crayfish Procambarus fallax
Guidelines
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.
Materials
UltraPure™ DEPC-treated WaterThermo FisherCatalog #10813012
TaqMan™ Environmental Master Mix 2.0 https://www.thermofisher.com/order/catalog/product/4396838#/4396838
Safety warnings
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.
DNA extraction
DNA extraction
2h 30m
2h 30m
A tissue of marbled crayfish was extracted with DNeasy blood and tissue extraction kit
The quality of DNA was checked by nanodrop.
Primers
| A | B | C | D | E | F | |
| Procambarus fallax | mtDNA-CO1 | 181 bp | Temp (C ) | Length | GC(%) | |
| Profal_COI_F01 | 5'-AGTTGAGAGGGGAGTAGGAAC-3 | 56.5 | 21 | 52.4 | ||
| Profal_COI_R01 | 5'-AGTTATACCAGCTGCCCGTA-3' | 57.4 | 20 | 50 | ||
| Profal_COI_P01 | 5'-FAM-AACTGTTTATCCTCCTTTAGCTTCTGC-BHQ1-3' | 62.6 | 27 | 40.7 |
2 µL Standard dilution
DNA of marbled crayfish was serially diluted from 1e2-1e-4 for qPCR experiment.
30m
PCR mixture
| A | B | C | D | |
| Stock solution | Working solution | Final concetration (μl) | ||
| TaqMan Environmental Mastermix 2 | 2X | 1X | 10 | |
| Forward primer | 10 μM | 0.4 μM | 1 | |
| Reverse primer | 10 μM | 0.4 μM | 1 | |
| TaqMan probe | 2.5 μM | 0.1 μM | 1 | |
| Internal control (IC) primer/probe mix | 1 | |||
| IC-DNA | 1 | |||
| Water | 8 | |||
| Template | 2 | |||
| Total | 25 |
2 µl of RNase/DNase free water was used for negative controls
PCR mixture can be lowered to 12 ul instead of 25 ul showed the same efficiency.
2h 30m
- °C Amplification conditions
| A | B | C | D | |
| Step | Time | Temp (℃) | ||
| Preheat | 5 min | 50 | ||
| Enzyme activation | 10 min | 95 | ||
| 50 cycles | Denaturation | 30 s | 95 | |
| Extention and Data collection | 1 min | 60 |
qPCR was performed in BioRad qPCR machine CFX96.
Expected result
Analysis of the results was done by CFX maestro software
Expected result
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 27±3 are within the normal range.
When amplifying 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.
Primer validation
DNA of related crayfish species were tested to ensure primers specificity.
| A | B | C | |
| Related species | Tested | Amplification | |
| Faxonius rusticus | Yes | No | |
| Faxonius virilis | Yes | No | |
| Faxonius immunis | Yes | No | |
| Faxonius juvenilis | Yes | No | |
| Pontastacus leptodactylus | Yes | No | |
| Astacus astacus | Yes | No |