Real-time quantitative loop-mediated isothermal amplification as a simple method for detecting white spot syndrome virus.

Mekata, T; Sudhakaran, R; Kono, T; Supamattaya, K; Linh, N T H; Sakai, M; Itami, T

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 degrees 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 (T(t)) using plasmid standards with high correlation coefficient (R(2) = 0.988). CONCLUSIONS Sensitivity analysis using 10-fold dilutions (equivalent to 35 ng microl(-1) to 35 ag microl(-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.