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Infectious diseases caused by <i>Streptococcus iniae</i> lead to massive death of fish, compose a serious threat to the global aquaculture industry, and constitute a risk to humans who deal with raw fish. In order to realize the early diagnosis of <i>S. iniae</i>, and control the outbreak and spread of disease, it is of great significance to establish fast, sensitive, and convenient detection methods for <i>S. iniae</i>. In the present study, two methods of real-time MIRA (multienzyme isothermal rapid amplification, MIRA) and MIRA-LFD (combining MIRA with lateral flow dipsticks (LFD)) for the <i>simA</i> gene of <i>S. iniae</i> were established, which could complete amplification at a constant temperature of 42 °C within 20 min. Real-time MIRA and MIRA-LFD assays showed high sensitivity (97 fg/μL or 7.6 × 10² CFU/mL), which were consistent with the sensitivity of real-time PCR and 10 times higher than that of PCR with strong specificity, repeatability simplicity, and rapidity for <i>S. iniae</i> originating from <i>Trachinotus ovatus</i>. In summary, real-time MIRA and MIRA-LFD provide effective ways for early diagnosis of <i>S. iniae</i> in aquaculture, especially for units in poor conditions.