Detection of Kudoa septempunctata18S Ribosomal DNA in Patient Fecal Samples from Novel Food-Borne Outbreaks Caused by Consumption of Raw Olive Flounder (Paralichthys olivaceus)

ABSTRACTKudoa septempunctatais a newly identified myxosporean parasite of olive flounder (Paralichthys olivaceus) and a suspected causative agent of several food-borne gastroenteritis outbreaks in Japan. Here, we report the detection of K. septempunctata18S ribosomal DNA in fecal samples of outbreak patients using an efficient method based on real-time PCR. We first performed a spiking experiment to assess whether our previously developed real-time PCR assay was applicable to detect K. septempunctatain feces. Simultaneously, we compared the relative extraction efficacy of K. septempunctataDNA using three commercial kits. Finally, our detection method was validated by testing 45 clinical samples obtained from 13 food-borne outbreaks associated with the consumption of raw flounder and 41 fecal samples from diarrhea patients epidemiologically unrelated to the ingestion of raw fish. We found that the FastDNA Spin Kit for Soil (MP Biomedicals) was the most efficient method for extracting K. septempunctataDNA from fecal samples. Using this kit, the detection limit of our real-time PCR assay was 1.6 × 101spores per g of feces, and positive results were obtained for 21 fecal and 2 vomitus samples obtained from the food-borne outbreaks. To our knowledge, this is the first report to describe the detection of K. septempunctataDNA in patient fecal samples. We anticipate that our detection method will be useful for confirming food-borne diseases caused by K. septempunctatain laboratory investigations.