title: Real-time quantitative loop-mediated isothermal amplification as a simple method for detecting white spot syndrome virus.
abstract: 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.
authors.affiliation: Interdisciplinary Graduate School of Agriculture and Engineering, University of Miyazaki, 1-1 Gakuen Kibanadai-nishi, Miyazaki, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Mekata
authors.forename: R
authors.initials: R
authors.lastname: Sudhakaran
authors.forename: T
authors.initials: T
authors.lastname: Kono
authors.forename: K
authors.initials: K
authors.lastname: Supamattaya
authors.forename: N T H
authors.initials: NT
authors.lastname: Linh
authors.forename: M
authors.initials: M
authors.lastname: Sakai
authors.forename: T
authors.initials: T
authors.lastname: Itami
mesh_terms: 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*
chemical_list: D017931:DNA Primers