Comparative evaluation of three real-time polymerase chain reaction assays to detect Myxobolus cerebralis.

Autor: Cavender W; Utah Division of Wildlife Resources, Aquatic Animal Health and Research Center, Logan, Utah, USA., Swan C; Utah Division of Wildlife Resources, Aquatic Animal Health and Research Center, Logan, Utah, USA., Wolf S; Utah Division of Wildlife Resources, Aquatic Animal Health and Research Center, Logan, Utah, USA., Van Vliet D; Utah Division of Wildlife Resources, Aquatic Animal Health and Research Center, Logan, Utah, USA., Johnson AA; Utah Division of Wildlife Resources, Aquatic Animal Health and Research Center, Logan, Utah, USA., Forest A; Utah Division of Wildlife Resources, Aquatic Animal Health and Research Center, Logan, Utah, USA., Shields R; Utah Division of Wildlife Resources, Aquatic Animal Health and Research Center, Logan, Utah, USA., Loch T; Department of Fisheries and Wildlife, Michigan State University, East Lansing, Michigan, USA.; Department of Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, Michigan, USA., Knupp C; Department of Fisheries and Wildlife, Michigan State University, East Lansing, Michigan, USA.; Department of Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, Michigan, USA., Drennan J; Colorado Parks and Wildlife, Aquatic Animal Health Laboratory, Brush, Colorado, USA., Glenney G; U.S. Fish and Wildlife Service, Lamar Fish Health Center, Lamar, Pennsylvania, USA., Hallett SL; Department of Microbiology, Oregon State University, Corvallis, Oregon, USA., Marcino J; Arizona Game and Fish Department, Fish Health Laboratory, Phoenix, Arizona, USA., Reed A; Oregon Department of Fish and Wildlife, Fish Health Services, Corvallis, Oregon, USA.
Jazyk: angličtina
Zdroj: Journal of aquatic animal health [J Aquat Anim Health] 2024 Sep; Vol. 36 (3), pp. 250-264. Date of Electronic Publication: 2024 Apr 15.
DOI: 10.1002/aah.10220
Abstrakt: Objective: We sought to evaluate accurate and reproducible detection of Myxobolus cerebralis (Mc), the causative agent of whirling disease, by using nested polymerase chain reaction (nPCR) and three previously established real-time quantitative PCR (qPCR) assays: K18S (Kelley 18S), C18S (Cavender 18S), and Hsp70 (heat shock protein 70). We used a "fit for purpose" approach combined with intra- and interlaboratory testing to identify a molecular testing method that would be equivalent to the currently accepted nPCR procedure for Mc.
Methods: Assay performance was compared using a combination of intra- and interlaboratory testing that used synthetic gBlocks along with naturally and experimentally infected fish tissue. North American isolates representing geographically distinct locations were also tested using all three assays.
Result: The K18S and C18S assays exhibited high assay sensitivity, intra- and interlaboratory repeatability of sample replicates, and reproducible identification of all test samples across multiple laboratories. In contrast, the Hsp70 assay failed to detect several positive samples at low DNA concentrations during intra- and interlaboratory testing. The K18S assay was the only procedure that demonstrated perfect detection accuracy when testing geographically distinct Mc isolates. Results demonstrated the K18S assay is robust under variable test conditions, is more accurate than the C18S and Hsp70 assays, and provides detection capabilities equivalent to those of the currently accepted nPCR confirmation assay "gold standard" that is described in the American Fisheries Society-Fish Health Section (AFS-FHS) Blue Book.
Conclusion: The "fit for purpose" approach and preliminary completion of the World Organization for Animal Health validation pathway demonstrate that the K18S assay provides an alternate method for Mc testing. This work provides the foundation for acceptance of the K18S assay into the AFS-FHS Blue Book as a standardized test procedure for Mc.
(© 2024 American Fisheries Society.)
Databáze: MEDLINE
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