Comparative evaluation of three real-time polymerase chain reaction assays to detect Myxobolus cerebralis.
Myxobolus cerebralis
limit of detection
real‐time PCR
Journal
Journal of aquatic animal health
ISSN: 1548-8667
Titre abrégé: J Aquat Anim Health
Pays: United States
ID NLM: 9884881
Informations de publication
Date de publication:
15 Apr 2024
15 Apr 2024
Historique:
revised:
05
03
2024
received:
26
12
2023
accepted:
06
03
2024
medline:
16
4
2024
pubmed:
16
4
2024
entrez:
16
4
2024
Statut:
aheadofprint
Résumé
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. 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. 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. 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.
Banques de données
RefSeq
['AF115253', 'EF370479.1', 'EF370480.1', 'EF370481.1']
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Subventions
Organisme : Great Lakes Fishery Commission
ID : 2022-CAV-441013
Informations de copyright
© 2024 American Fisheries Society.
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