Abstract
We designed a LAMP (loop-mediated isothermal amplificatio) primer set comprised with six primers from koi herpesvirus (KHV, CyHV-3) specific PCR amplicon (accession number: AF411803) and developed a rapid and sensitive detection method for KHV. The target sequence was amplified under isothermal condition at 65°C within 60 min, and the detection limit was at least six copies of the plasmid inserted KHV specific sequence. Cross-reactivity with other fish-pathogenic viruses and bacteria was not observed. The reaction was monitored in real-time by an increase in the turbidity of a large amount of by-product, pyrophosphate. As an alternative method, a visual detection at end-point of reaction could be achieved by an increase of turbidity in reaction mixture with naked eyes. The LAMP method was applicable to amplify the target sequence from crudely extracted samples of gills by a simple procedure.
