Development of conventional PCR and a Real-Time PCR assay to choose the most suitable method for residual DNA identification of Beluga, Huso huso, in concrete fish ponds

Document Type : Research Paper

Authors

1 Department of Biology, Faculty of Science, University of Guilan, Rasht, Iran

2 International Sturgeon Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Rasht, Iran

10.22124/cjes.2024.7984

Abstract

A unique research was developed to achieve an effective and efficient method for tracing and identifying Beluga sturgeon species, Huso huso using eDNA in concrete fish ponds. Sampling was performed from the water of the Beluga pond and fixed with precipitation premix solution. DNA was extracted from fixed water, and fin tissue of Beluga. Three short fragments were selected from a nuclear and two mitochondrial genes region of Beluga DNA. The specificity of primers was checked by conventional PCR for Beluga with fin tissue DNA samples. In this study, two conventional methods of molecular identification were performed on residual DNA samples of fish pond water and fin tissue, exhibiting that they were not suitable for measuring the residual DNA of Beluga in fish ponds due to the low rate of DNA. PCR amplification of sturgeon’s DNA of mitochondrial COΙ gene in third PCR primers was successful in low level of DNA extracted from water column and sediment by conventional and Quantitative PCR. The linear relationship between the threshold cycle (ct) value and Beluga DNA concentration was measured by the Mini-Barcoding quantitative Real-Time PCR. DNA samples of fish ponds generated a uniform curve in water column and sediment. In identifying residual DNA in the Picogram level of Beluga pond, SYBER Green Real-Time PCR method can be an acceptable method for barcoding with high efficiency compared to other methods such as conventional PCR method and non-invasive which is advantageous for the conservation of critically endangered sturgeons such as Beluga. This research results can also be used as a model for tracing fish species in rivers and seas.

Keywords

References

Barnes, MA & Turner, CR 2016, The ecology of environmental DNA and implications for conservation genetics. Conservation Genetics, 17: 1-17.
Bergman, PS, Schumer, G, Blankenship, S & Campbell, E 2016, Detection of adult green sturgeon using environmental DNA analysis. PLoS ONE, 11: e0153500.
Bronzi, P & Rosenthal, H 2014, Present and future sturgeon and caviar production and marketing: A global market overview. Journal of Applied Ichthyology, 30: 1536-1546.
Boscari, E, Vitulo, N, Ludwig, A, Caruso, C, Mugue, NS, Suciu, R, Onara, DF, Papetti, C, Marino, IA, Zane, L & Congiu, L 2017, Fast genetic identification of the Beluga sturgeon and its sought-after caviar to stem illegal trade. Food Control, 75: 145-152.
Deiner, K, Bik, HM, Mächler, E, Seymour, M, Lacoursière‐Roussel, A, Altermatt, F, Creer, S, Bista, I, Lodge, DM, De Vere, N & Pfrender, ME 2017, Environmental DNA metabarcoding: Transforming how we survey animal and plant communities. Molecular Ecology, 26: 5872-5895.
Doukakis, P, Pikitch, EK, Rothschild, A, DeSalle, R, Amato, G & Kolokotronis, SO 2012, Testing the effectiveness of an international conservation agreement: marketplace forensics and CITES caviar trade regulation. PLOS One, 7: e40907.
Goldberg, CS, Sepulveda, A, Ray, A, Baumgardt, J & Waits, LP 2013, Environmental DNA as a new method for early detection of New Zealand mud snails (Potamopyrgus antipodarum). Freshwater Science, 32: 792-800.
Jamshidi, S, Monsef Shokri, M & Hassanzadeh Saber, M 2021, Rapid detection of 4 species of Acipenser genus in the Caspian Sea Basin sturgeons and Siberian sturgeon using mitochondrial genome amplification method. Journal of Fisheries, 74: 351-362.
Janosik, AM, Whitaker, JM, VanTassel, NM & Rider, SJ 2021, Improved environmental DNA sampling scheme for Alabama sturgeon provides new insight into a species once presumed extinct. Journal of Applied Ichthyology, 37: 178-185.
Kubista, M, Andrade, JM, Bengtsson, M, Forootan, A, Jonák, J, Lind, K, Sindelka, R, Sjöback, R, Sjögreen, B, Strömbom, L & Ståhlberg, A 2006, The Real-Time polymerase chain reaction. Molecular Aspects of Medicine, 27: 95-125.
Ladell, BA, Walleser, LR, McCalla, SG, Erickson, RA & Amberg, JJ 2019, Ethanol and sodium acetate as a preservation method to delay degradation of environmental DNA. Conservation Genetics Resources, 11: 83-88.
Lawson Handley, L 2015, How will the ‘molecular revolution ‘contribute to biological recording. Biological Journal of the Linnean Society, 115: 750-766.
Lodge, DM, Williams, S, MacIsaac, HJ, Hayes, KR, Leung, B, Reichard, S, Mack, RN, Moyle, PB, Smith, M, Andow, DA & Carlton, JT 2006, Biological invasions: recommendations for US policy and management. Ecological Applications, 16: 2035-2054.
Mugue, NS, Barmintseva, AE, Rastorguev, SM, Mugue, VN & Barmintsev, VA 2008, Polymorphism of the mitochondrial DNA control region in eight sturgeon species and development of a system for DNA-based species identification. Russian Journal of Genetics, 44: 793-798.
Nissom, PM 2007, Specific detection of residual CHO host cell DNA by Real-Time PCR. Biologicals, 35: 211-215.
Plough, LV, Bunch, AJ, Lee, BB, Fitzgerald, CL, Stence, CP & Richardson, B 2021, Development and testing of an environmental DNA (eDNA) assay for endangered Atlantic sturgeon to assess its potential as a monitoring and management tool. Environmental DNA, 3: 800-814.
Rosenthal, H, Gessner, J & Bronzi, P 2014, Conclusions and recommendations of the 7th International Symposium on Sturgeons: Sturgeons, Science and Society at the cross-roads– Meeting the Challenges of the 21st Century. Journal of Applied Ichthyology, 30: 1105-1108.
Schenekar, T, Schletterer, M & Weiss, SJ 2020, Development of a TaqMan qPCR protocol for detecting Acipenser ruthenus in the Volga headwaters from eDNA samples. Conservation Genetics Resources, 12: 395-397.
Taberlet, P, Coissac, E, Hajibabaei, M & Rieseberg, LH 2012, Environmental DNA. Molecular Ecology, 21: 1789-1793.
Thomsen, PF & Willerslev, E 2015, Environmental DNA: An emerging tool in conservation for monitoring past and present biodiversity. Biological Conservation, 183: 4-18.
Torabi Jafroudi, H, Jamshidi, S, Talesh Sasani, S & Bani, A 2023, Molecular identification of Residual DNA separated from the Persian sturgeon (Acipenser persicus) for Modelling eDNA Evaluation in Aquatic Ecosystem. Journal of Genetic Resources, 9: 103-110.
Waraniak, JM, Blumstein, DM & Scribner, KT 2017, Barcoding PCR primers detect larval lake sturgeon (Acipenser fulvescens) in diets of piscine predators. Conservation Genetics Resources, 10: 259-268.
Yu, D, Shen, Z, Chang, T, Li, S & Liu, H 2021, Using environmental DNA methods to improve detectability in an endangered sturgeon (Acipenser sinensis) monitoring program. BMC Ecology and Evolution, 21: 1-11.
Caspian Journal of Environmental Sciences
Volume 22, Issue 4
October 2024
Pages 955-961

Files

Share

How to cite

Statistics