Feb 03, 2025

Public workspaceDetection of marbled crayfish Procambarus fallax

DOI
https://dx.doi.org/10.17504/protocols.io.q26g78199lwz/v1
  • Alexander Eiler1,
  • Eivind Stensrud1,
  • Omneya Osman2
  • 1eDNA solutions AB;
  • 2IVL Swedish research Institute
Omneya Osman
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DOI: https://dx.doi.org/10.17504/protocols.io.q26g78199lwz/v1
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
Keywords: marbled crayfish procambarus, procambarus, taqman qpcr, detection
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
Interbal control http://www.primerdesign.co.uk/assets/files/internal_control_handbook_dna.pdf?timestamp=1504081027
Troubleshooting
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
2h 30m

A tissue of marbled crayfish was extracted with DNeasy blood and tissue extraction kit
https://www.qiagen.com/us/shop/pcr/dneasy-blood-and-tissue-kit/
The quality of DNA was checked by nanodrop.

Primers

ABCDEF
Procambarus fallaxmtDNA-CO1181 bpTemp (C )LengthGC(%)
Profal_COI_F015'-AGTTGAGAGGGGAGTAGGAAC-356.52152.4
Profal_COI_R015'-AGTTATACCAGCTGCCCGTA-3'57.42050
Profal_COI_P015'-FAM-AACTGTTTATCCTCCTTTAGCTTCTGC-BHQ1-3'62.62740.7

Amount2 µL Standard dilution

DNA of marbled crayfish was serially diluted from 1e2-1e-4 for qPCR experiment.
30m
PCR mixture
ABCD
Stock solutionWorking solutionFinal concetration (μl)
TaqMan Environmental Mastermix 22X1X10
Forward primer10 μM0.4 μM1
Reverse primer10 μM0.4 μM1
TaqMan probe2.5 μM0.1 μM1
Internal control (IC) primer/probe mix1
IC-DNA1
Water8
Template2
Total25
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
Temperature- °C Amplification conditions

ABCD
StepTimeTemp (℃)
Preheat5 min50
Enzyme activation10 min95
50 cyclesDenaturation30 s95
Extention and Data collection1 min60

qPCR was performed in BioRad qPCR machine CFX96.

Expected result



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.
ABC
Related speciesTestedAmplification
Faxonius rusticus YesNo
Faxonius virilis YesNo
Faxonius immunis YesNo
Faxonius juvenilis YesNo
Pontastacus leptodactylusYesNo
Astacus astacusYesNo