Development of a quantitative polymerase chain reaction assay for detection of Kudoa septempunctata in olive flounder (Paralichthys olivaceus)

Abstract

Kudoa septempunctata is a newly identified myxosporean parasite that infects the trunk muscles of olive flounder (Paralichthys olivaceus) and a causative agent of the increasing number of foodborne gastroenteritis outbreaks with unknown etiology which have occurred in Japan over the last few years. Here, we developed a quantitative polymerase chain reaction (QPCR) assay for the detection of K. septempunctata 18S rDNA in olive flounder muscle tissue samples. Additionally, we compared the relative efficacy of four DNA extraction methods, including two commercial kits, and assessed intrafish variability in the distribution of K. septempunctata spores in flounder using this QPCR method in order to establish a more accurate quantitative measurement. Our QPCR assay displayed high sensitivity, specificity, and reproducibility, and had good correlation with a microscopic detection method. Our data also indicated that the DNeasy® Blood & Tissue Kit was more efficient method for the extraction of K. septempunctata DNA than the three other methods (heating, alkaline lysis, and FastDNA® SPIN Kit method). We believe that our method would be useful for investigating foodborne outbreaks caused by K. septempunctata and for the monitoring and quantification of this parasite in retail or aquacultured olive flounders to prevent such outbreaks.