Abstract
A probe-based quantitative real-time PCR assay was developed to detect Tetracapsuloides bryosalmonae, which causes proliferative kidney disease in salmonid fish, in kidney tissue and environmental DNA (eDNA) water samples. The limits of detection and quantification were 7 and 100 DNA copies for calibration standards and T. bryosalmonae was reliably detected down to 100 copies in tissue and eDNA samples. The assay presented here is a highly sensitive and quantitative tool for detecting T. bryosalmonae with potential applications for tissue diagnostics and environmental detection.
This is a preview of subscription content, log in via an institution to check access.
References
Canning E, Curry A, Feist S, Longshaw M, Okamura B (1999) Tetracapsula bryosalmonae n. sp. for PKX organism, the cause of PKD in salmonid fish. Bull Eur Assoc Fish Pathol 19:203–206
Carim K, Wilcox T, Young M, Schwartz M, McKelvey K (2015) Protocol for collecting eDNA samples from streams. USDA Forest Service, Rocky Mountain Research Station, Missoula, Montana. http://www.fs.fed.us/research/geno mics-center/docs/edna/edna-protocol.pdf
Forootan A, Sjӧback R, Bjӧrkman J, Sjӧgreen B, Linz L, Kubista M (2017) Methods to determine limit of detection and limit of quantification in quantitative real-time PCR (qPCR). Biomol Detect Quantif. doi:10.1016/j.bdq.2017.04.001
Hallett SL, Bartholomew JL (2006) Application of a real-time PCR assay to detect and quantify the myxozoan parasite Ceratomyxa shasta in river water samples. Dis Aquat Org 71:109–118. doi:10.3354/dao071109
Kent M, Khattra J, Hervio D, Devlin R (1998) Ribosomal DNA sequence analysis of isolates of the PKX myxosporean and their relationship to members of the genus Sphaerospora. J Aquat Anim Health 10:12–21
Okamura B, Hartikainen H, Schmidt-Posthaus H, Wahli T (2011) Life cycle complexity, environmental change and the emerging status of salmonid proliferative kidney disease. Freshw Biol 56:735–753. doi:10.1111/j.1365-2427.2010.02465.x
Ruijter JM, Ramakers C, Hoogaars WMH, Karlen Y, Bakker O, van den Hoff MJB, Moorman AFM (2009) Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Res 37:e45. doi:10.1093/nar/gkp045
Ruijter JM, Villalba AR, Hellemans J, Untergasser A, van den Hoff MJ (2015) Removal of between-run variation in a multi-plate qPCR experiment. Biomol Detect Quantif 5:10–14. doi:10.1016/j.bdq.2015.07.001
Taberlet P, Coissac E, Hajibabaei M, Rieseberg LH (2012) Environmental DNA. Mol Ecol 21:1789–1793. doi:10.1111/j.1365-294X.2012.05542.x
Tuomi JM, Voorbraak F, Jones DL, Ruijter JM (2010) Bias in the Cq value observed with hydrolysis probe based quantitative PCR can be corrected with the estimated PCR efficiency value. Methods 50:313–322. doi:10.1016/j.ymeth.2010.02.003
Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden TL (2012) Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinform 13:134. doi:10.1186/1471-2105-13-134
Acknowledgements
The work presented here was funded by the United States Fish and Wildlife Service Region 6 Fish Health Center and the United States Geological Survey Invasive Species Program. A special thanks to Jamie Brusa for help in the field. Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the United States Government. The findings and conclusions in this article are those of the authors and do not necessarily represent the views of the United States Fish and Wildlife Service.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Disclosure
The authors have nothing to disclose.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Hutchins, P.R., Sepulveda, A.J., Martin, R.M. et al. A probe-based quantitative PCR assay for detecting Tetracapsuloides bryosalmonae in fish tissue and environmental DNA water samples. Conservation Genet Resour 10, 317–319 (2018). https://doi.org/10.1007/s12686-017-0812-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12686-017-0812-3