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Aims:  White spot syndrome virus (WSSV) continues to be the most pathogenic virus among the crustacean aquaculture causing mass mortality. In the present study, we established a one‐step, single tube, real‐time accelerated loop‐mediated isothermal amplification (real‐time LAMP) for quantitative detection of WSSV. Materials and Methods:  A set of six specially designed primers that recognize eight distinct sequences of the target. The whole process can be completed in 1 h under isothermal conditions at 63°C. Detection and quantification can be achieved by real‐time monitoring in an inexpensive turbidimeter based on threshold time required for turbidity in the LAMP reaction. A standard curve was constructed by plotting viral titre against the threshold time (Tt) using plasmid standards with high correlation coefficient (R2 = 0·988). Conclusions:  Sensitivity analysis using 10‐fold dilutions (equivalent to 35 ng μl−1 to 35 ag μl−1) of plasmid standards revealed this method is capable of detecting upto 100 copies of template DNA. Cross‐reactivity analysis with DNA/cDNA of IHHNV, TSV, YHV‐infected and healthy shrimp showed this method is highly specific for quantitative detection of WSSV. Significance and Impact of the Study:  WSSV real‐time LAMP assay appears to be precise, accurate and a valuable tool for the detection and quantification of WSSV in large field samples and epidemiological studies.

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ABSTRACT-A one-step, single tube, real-time loop-mediated isothermal amplification (real-time LAMP) assay was developed to detect a non-structural protein encoding gene of infectious hypodermal and haematopoietic necrosis virus (IHHNV). The real-time LAMP method for IHHNV is simple and rapid with specific amplification within 60 min at 63°C employing four primers. This method requires less time than the PCR method and is specific for IHHNV when analysed with PRDV (= WSSV), YHV and TSV DNA/cDNA, and healthy shrimp DNA. Sensitivity analysis revealed this method is capable of detecting as few as 10 2 -10 3 copies/µL, suggesting it can be used as an alternative quantitative detection method for IHHNV in diagnostic laboratories.