Development of Quantitative Real-time PCR and Loop-mediated Isothermal Amplification (LAMP) Assays for Detection of Microsporidium seriolae

Beko disease, caused by the Microsporidium seriolae infection, has been a problem in yellowtail aquaculture in western Japan. In recent years, severe cases of this disease have been confirmed, resulting in a significant decrease in product value due to mass mortality and residual cysts. Only polymerase chain reaction (PCR) assays have been reported so far as a detection method for the disease. In this study, quantitative real-time PCR (qPCR) and loop-mediated isothermal amplification (LAMP) methods have been optimized for M. seriolae detection in order to establish a more sensitive and rapid diagnosis. Target regions for each detection method were selected based on the nucleotide sequences obtained by the gene analysis of cysts in diseased fish. Primer sets for the qPCR and LAMP methods were designed, and the gene amplification efficiency of each method was evaluated. The results showed that the newly developed qPCR method could detect 1.4 copies of the target gene, and the LAMP method detected 100 copies within 15 minutes. In this study, the newly developed qPCR and LAMP assays were shown to be rapid and highly sensitive methods for quantitative detection of M. seriolae.