Sep 05, 2024
Probe qPCR assay for Charybdis japonicum
This protocol is a draft, published without a DOI.
- Jackson Treece1,
- Gert-Jan Jeunen1,
- Jo-Ann Stanton1,
- Neil Gemmell1
- 1University of Otago
External link: https://www.biosecurity-toolbox.org.nz/
Protocol Citation: Jackson Treece, Gert-Jan Jeunen, Jo-Ann Stanton, Neil Gemmell 2024. Probe qPCR assay for Charybdis japonicum . protocols.io https://protocols.io/view/probe-qpcr-assay-for-charybdis-japonicum-dki64uhe
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: September 02, 2024
Last Modified: September 05, 2024
Protocol Integer ID: 106814
Keywords: charybdis, charybdis japonicum, invasive crustacean, invasive crab, charybdis qpcr, charybdis japonicum qpcr, biosecurity toolbox, marine biosecurity, marine biosecurity toolbox, new zealand native crab species, native crab species, invasive crustacean, asian paddle crab, using environmental dna sampling, environmental dna sampling, charybdis japonicum, environmental dna, charybdis japonica, threat to local native species, qpcr, species specificity, local native species, further challenge with local species, genomic dna input, based qpcr, local species
Funders Acknowledgements:
New Zealand Ministry for Business, Innovation and Employment
Grant ID: CAWX1904
Abstract
Charybdis japonica or the Asian Paddle Crab is a non-native crab species to Aotearoa, New Zealand. It was first detected in 2000 and has since spread along the eastern Northland coast where it is a threat to local native species. This test was developed to aid in detecting the invasive crustacean's presence using environmental DNA sampling.
The probe based qPCR assay targets a 208bp portion of the COX-1 gene with an indicative limit of detection of 1.0E-4 ng/µl genomic DNA input or 92 molecules/µl with a synthetic standard (IDT gBlock). Species specificity was successfully challenged against a panel of 14 New Zealand native crab species. Further challenge with local species is recommended.
Troubleshooting
Materials
Meridian Bioscience SensiFAST Probe Lo-ROX Mix (2x)
PCR quality water
Template DNA
Oligonucleotides (10uM concentration):
Forward primer: Cj_1295F ATGGCATGCCTCGTCGTTAT
Reverse primer: Cj_1498R CTGCAGGTGGGTAAGAGTGG
FAM Probe: Cj_1323P CCCCGATGCATACACTACGT
Master Mix
10.0 µl SensiFAST Probe 2x Mix
0.8 µl 10µM Forward primer
0.8 µl 10µM Reverse primer
0.2 µl 10µM FAM Probe
6.2 µl PCR Water
2.0 µl Template
20µl total reaction volume
Thermal Conditions
95°C - 3 minutes
95°C - 15 seconds
60°C - 30 seconds x45 cycles
QuantStudio 3 (ThermoFisher Scientific) used during assay development.
Control Sequence
Sythesized positive control sequence (IDT gBlocks)
tattactgccattcttctactcctctcgcttcctgtgttagctggagctattactatattattaacagaccgaaacttaaatacttcattcttcgaccctgctgggggtggggatcctgttctttatcaacacttgttttgattctttggacatcctgaagtttatattttaatcttacctgccttcggaataatttcacatattgtcagacaagaatctggtaaaaaggaatcatttggtactttgggtataatttatgctataatggctattggcattttaggctttattgtatgagctcatcacatgtttacagttggaatagatgtagatactcgagcatattttacatcagctactataattattgctgtgcctactggaattaaaatttttagatgacttagaactcttcacggcactcaaattaactatagaccttctatattatgggccctaggatttattttcttattcacagtaggaggtcttacaggagtagttttagcaaactcttccattgatattattcttcacgacacatactatgtggtagcccactttcattacgttctttctataggagctgtatttggaattttcgccggcattgcccactgattccccttatttaccggactttcattaaatcctaaatgaataaaaattcattttactattatatttattggtgtaaatattactttctttcctcaacactttttaggactaaatggcatgcctcgtcgttattctgactaccccgatgcatacactacgtgaaatattgtctcttctataggatcaatagtatcacttattgctatactaatttttataattgtaatttgagaagcacttatctcaaatcgacctgttctattttctcctttccttccttcttctattgaatgaaaccactcttacccacctgcagatcactcatacatagaaatccccctaatctctaacttctaaaatggcagatagatgtatagg
Species for Assay Development and Specificity Testing
Many thanks to the National Institute of Water and Atmospheric Research (NIWA), the Marine Invasives Taxonomic Service (MITS) and Deakin University for contributions from their invertebrate tissue collection. Donation ID: 2023-032 D
| Species | Accession | |
| Austrohelices crassa | MITS_74324 | |
| Carcinus maenas | N/A (Deakin) | |
| Charybdis japonica | MITS_70207 | |
| Elamena producta | NIWA_89937 | |
| Halicarcinus cookii | NIWA_81032 | |
| Halicarcinus whitei | NIWA_147705 | |
| Heterozius rotundifrons | NIWA_163347 | |
| Liocarcinus corrugatus | NIWA_73086 | |
| Nectocarcinus antarcticus | NIWA_74132 | |
| Notomithrax minor | NIWA_92580 | |
| Notomithrax peronii | NIWA_72993 | |
| Ovalipes catharus | NIWA_60579 | |
| Pilumnus lumpinus | NIWA_75667 | |
| Pilumnus novaezealandiae | NIWA_92423 | |
| Pinnotheres atrinicola | NIWA_74906 |