New validated rapid screening methods for identifying Kudoa septempunctata in olive flounder (Paralichthys olivaceus)

Kudoa septempunctata is a newly identified causative agent of foodborne diseases associated with consuming raw olive flounder. Qualitative PCR and quantitative real-time PCR have been used as notification methods to identify K. septempunctata in Japan. However, these methods require expensive equipment and are time-consuming (2-3 h for screening). To address these problems, in this study, we developed new rapid and simple methods using real-time loop-mediated isothermal amplification (LAMP) and nucleic acid sequence based amplification-nucleic acid chromatography (NASBA-NAC). Using these methods, the total procedure required approximately 45 min and did not require any expensive equipment. With regard to validating these new methods in comparison with the notification methods used in Japan, we performed an inter-laboratory study of 5 laboratories using samples that included olive flounders infected with 4 different amounts of K. septempunctata. These results demonstrated that the sensitivity of NASBA-NAC was equivalent to that of qualitative PCR, and that the sensitivity of real-time LAMP was equivalent to that of quantitative real-time PCR, which indicated that these new methods were acceptable screening methods for identifying K. septempunctata.