넙치 주요 세균성 병원체 4종의 동시 검출을 위한 Multiplex PCR 기법 개발
Development of Multiplex PCR for Detecting Four Bacterial Pathogens in Olive flounder (Paralichthys olivaceus)
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Bacterial diseases pose a major threat to marine aquaculture, causing serious losses in olive flounder (Paralichthys olivaceus). Four major pathogens－Tenacibaculum maritimum, Edwardsiella piscicida, Vibrio harveyi, and Streptococcus parauberis－are associated with severe mortality and production decline. Conventional diagnostic methods such as culture, serological assays, and singleplex PCR are often slow, cross-reactive, and unsuitable for mixed infections. Here, we developed a multiplex PCR (mPCR) assay for simultaneous detection of four pathogens. Species-specific primers were de- signed or adapted, and assay conditions were optimized for clear amplicon separation. The mPCR showed high specificity without cross-reactivity to 15 related bacteria and detected as low as 5.0× 10^1 copies/µL for T. maritimum, and 10^2 copies/µL for the others. It also identified all four pathogens in spiked olive flounder tissues (spleen and kidney) and environmental samples (seawater and biofilm) at 10² cells/g or 10² cells/L. This mPCR provides a rapid, sensitive, and cost-effective diagnostic platform for early detection and on-farm monitoring of bacterial diseases in olive flounder aquaculture.
Abstract
Bacterial diseases pose a major threat to marine aquaculture, causing serious losses in olive flounder (Paralichthys olivaceus). Four major pathogens－Tenacibaculum maritimum, Edwardsiella piscicida, Vibrio harveyi, and Streptococcus parauberis－are associated with severe mortality and production decline. Conventional diagnostic methods such as culture, serological assays, and singleplex PCR are often slow, cross-reactive, and unsuitable for mixed infections. Here, we developed a multiplex PCR (mPCR) assay for simultaneous detection of four pathogens. Species-specific primers were de- signed or adapted, and assay conditions were optimized for clear amplicon separation. The mPCR showed high specificity without cross-reactivity to 15 related bacteria and detected as low as 5.0× 10^1 copies/µL for T. maritimum, and 10^2 copies/µL for the others. It also identified all four pathogens in spiked olive flounder tissues (spleen and kidney) and environmental samples (seawater and biofilm) at 10² cells/g or 10² cells/L. This mPCR provides a rapid, sensitive, and cost-effective diagnostic platform for early detection and on-farm monitoring of bacterial diseases in olive flounder aquaculture.