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Title (en)
Development of a rapid and sensitive real-time diagnostic assay to detect and quantify Aphanomyces invadans, the causative agent of epizootic ulcerative syndrome
Language
English
Description (en)
The oomycete Aphanomyces invadans causes epizootic ulcerative syndrome (EUS), a World Organization for Animal Health (WOAH)-listed disease that has seriously impacted a wide range of fish worldwide. Currently, only three conventional polymerase chain reaction (PCR) assays are recommended for the detection of A. invadans. The robust quantitative PCR (qPCR) assay has recently become more important due to its highly accurate nature and the applicability of qPCR-based environmental DNA (eDNA) detection in the monitoring of pathogens in aquatic environments. Therefore, in this study, we developed a novel TaqMan probe-based qPCR method to sensitively and quantitatively detect A. invadans. The assay limit of detection was determined using 10-fold serial dilutions of linearized A. invadans plasmid. Assay sensitivity was assessed in the presence of interfering substances and compared to three WOAH-listed primers using the mycelia and zoospores of A. invadans with and without fish muscle tissue. The assay specificity was also theoretically and experimentally assessed against other oomycetes, fish muscle tissue, and water samples. The assay's repeatability and reproducibility were determined. In this study, the limit of detection of the developed assay was 7.24 copies of A. invadans genomic DNA per reaction (95% confidence interval (CI): 2.75 to 19.05 copies/reaction). The assay showed the same sensitivity in the presence of other substances. Compared to the WOAH-recommended PCR assays, this assay had 10-times higher sensitivity for all tested samples. There were no cross-reactions with other closely related oomycetes, fish muscle, or water samples, indicating that the assay was highly specific for A. invadans. The repeatability and reproducibility tests showed little variation, ranging from 0.1-0.9% and 0.04-1.1%, respectively, indicating the high consistency, repeatability, and reliability of the developed assay. This highly rapid, sensitive, specific, and consistent EUS qPCR assay would be of importance in transboundary disease management and the monitoring of pathogens in aquatic environments.
Keywords (en)
Pcr; Identification
DOI
10.1371/journal.pone.0286553
Author of the digital object
Diem Tho Ho  (Pukyong National University)
Do-Hyung Kim  (Pukyong National University)
Ki Hong Kim  (Pukyong National University)
Chan-Il Park  (Gyeongsang National University)
Wi-Sik Kim  (Chonnam National University)
Neeraj Sood  (ICAR-National Bureau of Fish Genetic Resources)
P. K. Pradhan  (ICAR-National Bureau of Fish Genetic Resources)
El-Matbouli Mansour  (University of Veterinary Medicine Vienna)
MinJi Sung  (Pukyong National University)
Dongbin Yun  (Bioneer Corporation)
Nameun Kim  (Pukyong National University)
Yoonhang Lee  (Pukyong National University)
Format
application/pdf
Size
659.9 kB
Licence Selected
CC BY 4.0 International
Type of publication
Article
Name of Publication (en)
PloS one
Pages or Volume
13
Volume
18
Number
6
Publisher
Public Library of Science
Publication Date
2023