Development of a Quantitative PCR Assay for Four Salmon Species Inhabiting the Yangyangnamdae River Using Environmental DNA
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Target Species and Molecular Assay Development
2.2. Water Sample Collection and Environmental DNA Extraction
2.3. Establishment of Quantitative PCR Assay
2.4. Inhibition Test
2.5. Data Analysis
3. Results
3.1. Profile and Performance of Primers and Probes
3.2. Inhibition in Environmental DNA Samples
3.3. Environmental DNA Profiles in Field Testing
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Appendix A
Species | GenBank Accession Number |
---|---|
Oncorhynchus keta | MN011557, MN011565, MN011555, MN011556, MN011562, MN011567, MK991795, MK991796, MK991797, MN011560, MN011558, JX960808, KU872716, MN011553, MN011561, MN011552, MN011564, KR778826, KR778827, KR778832, MN011563, MK991794, MN011554, MN011559, KR778834, NC_017838, KR778839, KR778838, KR778836, KR778845, KR778848, KR778849, KR778831, KR778833, KR778828, MN011566, KR778837, AF125212, FJ435616, FJ435617 |
Oncorhynchus kisutch | KU761856, JX185441, JX185442, KU761856, NC_009263, JX960809, MF621749, KP671851, JX960810 |
Oncorhynchus masou | JX960818, NC_008746, LC098718, FJ435612, NC_008747, FJ435611, NC_008745, JX960811, KY250421, LC098722, NC_009262, LC098720, LC098719 |
Oncorhynchus mykiss | AF125208, FJ435586, JX960814, KP013084, KU872710, AY032629, KU761858, AY032632, AY587174, AY587168, AY587172, AY587176, AF125209, MF621750, JX960815, DQ288271, AY032631, AY587169, FJ435599, NC_026537, FJ435595, AY587183, FJ435589, NC_001717, KP085590 |
O. keta | O. masou | O. mykiss | O. kisutch | ||
---|---|---|---|---|---|
qPCR | Luna® Universal Probe qPCR Master Mix (#M3004, New England Biolabs, Ipswich, MA, USA) | 10 µL | |||
Forward primer | 0.4 µM | 0.4 µM | 0.4 µM | 0.5 µM | |
Reverse primer | 0.4 µM | 0.4 µM | 0.4 µM | 0.5 µM | |
Probe | 0.2 µM | 0.2 µM | 0.2 µM | 0.25 µM | |
eDNA | 2 µL | ||||
ddH2O | Up to 20 µL | ||||
qPCR cycles | Initial denaturation | 95 °C, 5 min | |||
Denaturation | 95 °C, 20 s | ||||
Annealing | 63.6 °C, 20 s | 63.6 °C, 20 s | 64.9 °C, 20 s | 66.0 °C, 20 s | |
Extension | 72 °C, 20 s | ||||
Cycles | 40 |
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Species | Forward Primer (5′–3′) Reverse Primer (5′–3′) Probe (5′–3′) | Size (bp) |
---|---|---|
Oncorhynchus keta | CTACGGCTGACTAATTCGGAACATCCAC TCCTCACGGGAGGACGTAGCCC FAM-CGCCCGGGGACTTTATTACGGATCCTACCT-BHQ | 187 |
Oncorhynchus kisutch | TTACACACCTCCAAACAACGAGGACTG TTGGCCGATAATGATGAATGGGTGTTCC FAM-CCCAATTCCTATTCTGGGCCTTGGTGGCG-BHQ | 138 |
Oncorhynchus masou | GGGTTCTCTGTCGACAACGCCAC CTAAGGATGTTAGACAGAGAAGTATAGCTG FAM-CGTCATTACAGCTGCTGCAATCCTCCACCT-BHQ | 229 |
Oncorhynchus mykiss | GAGGACTTTACTACGGCTCGTACCTC GTTAGAGTGGCGTTGTCAACGGAGAAG FAM-CTGCCTTTGTAGGCTACGTCCTCCCGTGAG-BHQ | 229 |
Zenarchopterus dispar1 | CAGCAGCTATAAACGCATGAATTACAGG TTTTTGTCAGGTTGAGAGAATGAGTCCG | 188 |
Species | LOD (Copy Numbers) | LOQ (Copy Numbers) | Efficiency (%) |
---|---|---|---|
Oncorhynchus keta | 4.11 | 30 | 96.26 |
Oncorhynchus kisutch | 8.44 | 45 | 95.15 |
Oncorhynchus masou | 4.65 | 81 | 100.67 |
Oncorhynchus mykiss | 10.38 | 34 | 106.33 |
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Fu'adil Amin, M.H.; Lee, J.-H.; Kim, A.R.; Kim, J.-K.; Lee, C.-I.; Kim, H.-W. Development of a Quantitative PCR Assay for Four Salmon Species Inhabiting the Yangyangnamdae River Using Environmental DNA. Biology 2021, 10, 899. https://doi.org/10.3390/biology10090899
Fu'adil Amin MH, Lee J-H, Kim AR, Kim J-K, Lee C-I, Kim H-W. Development of a Quantitative PCR Assay for Four Salmon Species Inhabiting the Yangyangnamdae River Using Environmental DNA. Biology. 2021; 10(9):899. https://doi.org/10.3390/biology10090899
Chicago/Turabian StyleFu'adil Amin, Muhammad Hilman, Ji-Hyun Lee, Ah Ran Kim, Ju-Kyoung Kim, Chung-Il Lee, and Hyun-Woo Kim. 2021. "Development of a Quantitative PCR Assay for Four Salmon Species Inhabiting the Yangyangnamdae River Using Environmental DNA" Biology 10, no. 9: 899. https://doi.org/10.3390/biology10090899
APA StyleFu'adil Amin, M. H., Lee, J. -H., Kim, A. R., Kim, J. -K., Lee, C. -I., & Kim, H. -W. (2021). Development of a Quantitative PCR Assay for Four Salmon Species Inhabiting the Yangyangnamdae River Using Environmental DNA. Biology, 10(9), 899. https://doi.org/10.3390/biology10090899