Point-of-care testing based on colorimetric loop-mediated isothermal amplification coupled with a nondestructive device for rapid detection of Kudoa septempunctata in live olive flounder

Abstract

Kudoa septempunctata, a myxosporean parasite newly identified in 2010, was first detected in the trunk muscle of an aquacultured olive flounder imported from Korea to Japan. Since 2003, foodborne disease outbreaks in Japan have been increasing, often linked to the ingestion of raw olive flounder. These illnesses, characterized by diarrhea and emesis, typically occur within 2–20 h after consuming raw olive flounder. Recent studies on the etiological agents responsible for these illnesses and animal experiments have implicated K. septempunctata as a potential causative agent of these novel foodborne disease outbreaks. Consequently, a rapid, sensitive, and convenient method for detecting K. septempunctata is required. This study presents a colorimetric loop-mediated isothermal amplification (LAMP) assay developed using a molecular beacon (MB) and horseradish peroxidase-mimicking DNAzyme for the rapid and sensitive detection of K. septempunctata in the muscle of olive flounder. Three pairs of primers targeting the 28S rDNA gene and an MB were designed and synthesized. The colorimetric LAMP assay was optimized by determining key factors such as MB concentration, incubation temperature, and time. Its specificity was investigated, and the method was validated using contaminated muscle samples of olive flounder (101–106 spores/g). The cutoff value of the colorimetric LAMP assay, optimized at 57.5 °C, was 1 × 101 spores/g, confirming its specificity to K. septempunctata. The developed assay can be completed within 1 h. Overall, this study demonstrates that the developed K. septempunctata-specific LAMP assay shows promise as a point-of-care molecular diagnostic technology, as it does not require expensive instruments such as a thermocycler or detector. © 2024