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


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.


CONCLUSIONS
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.
Mekata
T
T
Interdisciplinary Graduate School of Agriculture and Engineering, University of Miyazaki, 1-1 Gakuen Kibanadai-nishi, Miyazaki, Japan.
Sudhakaran
R
R
Kono
T
T
Supamattaya
K
K
Linh
N T H
NT
Sakai
M
M
Itami
T
T
D000818:Animals; D003445:Crustacea / Q000821:virology*; D017931:DNA Primers / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012680:Sensitivity and Specificity; D014777:Virus Diseases / Q000175:diagnosis / Q000662:veterinary*; D046848:White spot syndrome virus 1 / Q000302:isolation & purification*
D017931:DNA Primers