Premium
Real‐time quantitative loop‐mediated isothermal amplification as a simple method for detecting white spot syndrome virus
Author(s) -
Mekata T.,
Sudhakaran R.,
Kono T.,
Supamattaya K.,
Linh N.T.H.,
Sakai M.,
Itami T.
Publication year - 2009
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1111/j.1472-765x.2008.02479.x
Subject(s) - loop mediated isothermal amplification , white spot syndrome , biology , plasmid , real time polymerase chain reaction , virus , virology , shrimp , standard curve , complementary dna , serial dilution , microbiology and biotechnology , dna , genetics , gene , medicine , biochemistry , alternative medicine , pathology , fishery
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 ( T t ) using plasmid standards with high correlation coefficient ( R 2 = 0·988). Conclusions: Sensitivity analysis using 10‐fold dilutions (equivalent to 35 ng μ l −1 to 35 ag μ l −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.