Titel (eng)

Development of a rapid and sensitive real-time diagnostic assay to detect and quantify Aphanomyces invadans, the causative agent of epizootic ulcerative syndrome

Autor*in

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

Verlag

Public Library of Science

Beschreibung (eng)

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.

Sprache des Objekts

Englisch

Datum

2023

Rechte

Dieses Werk bzw. dieser Inhalt steht unter einer
CC BY 4.0 - Creative Commons Namensnennung 4.0 International Lizenz.

CC BY 4.0 International

http://creativecommons.org/licenses/by/4.0/

Klassifikation

Pcr; Identification

Mitglied in der/den Collection(s) (1)