Primary Establishment of LAMP Method for Cyprinid Herpesvirus 2 Detection

A new loop-mediated isothermal amplification (LAMP) method for the detection of Cyprinid herpesvirus 2 (CyHV-2) was developed. Following the synthesis in vitro of a 413 bp DNA fragment of CyHV2 DNA-dependent DNA polymerase gene by overlap extension PCR using 11 overlapping oligo primers designed according to the gensome sequences information of CyHV2 published in GenBank, a set of 6 LAMP primers were designed based on the synthesized DNA fragment, and the LAMP assay was primarily established after preparation of target gene fragments, optimization of reaction conditions, products identification, sensitivity and specificity tests. Results showed the developed LAMP assay was a rapid, specific and sensitive method for CyHV2 detection, with a ladder-like pattern of amplification bands from about 177 bp incubation at 64°C for 30 min by using agarose gel electrophoresis. The detection limit could reach about 15 copies of cloned viral genomic fragments, and no cross reaction with Aeromonas veronii, Aeromonas hydrophila, Pseudomonas fluorescens and Streptococcus common pathogens isolated from fish detection by this approach.