Comparative evaluation of three real-time polymerase chain reaction assays to detect Myxobolus cerebralis
Authors: Cavender Wade , Swan Christine , Wolf Skylar , Van Vliet Danielle , Johnson Alison Aceves , Forest Anna , Shields Robert , Loch Thomas , Knupp Christopher , Drennan John , Glenney Gavin , Hallett Sascha L. , Marcino Joe , Reed Aimee 
Published: April 2024

Abstract

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.
Abstract
Impact statement The myxozoan parasite Myxobolus cerebralis (Mc) can cause whirling disease in salmonids, which can negatively impact hatchery and wild populations. Rapid, sensitive detection of developmental stages is important to identify Mc through monitoring and testing of presumptive findings. The current “gold standard” for molecular testing is nested PCR, which requires two consecutive reactions and visualization of amplicons using gel electrophoresis. Alternatively, three previously published real-time quantitative PCR (qPCR) assays offer a faster, more sensitive approach for Mc testing. During intra- and interlaboratory comparisons, we evaluated these qPCR assays using a range of fish tissue and gBlocks. 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 molecular testing procedure for Mc.