A loop-mediated isothermal amplification assay for rapid detection of cyprinid herpesvirus 2 in gibel carp (Carassius auratus gibelio)

ScientificWorldJournal. 2014 Jan 19:2014:716413. doi: 10.1155/2014/716413. eCollection 2014.

Abstract

A rapid and sensitive loop-mediated isothermal amplification (LAMP) assay for Cyprinid herpesvirus 2 (CyHV-2) detection in gibel carp was developed. Following cloning and sequencing of the putative DNA helicase gene of CyHV-2 isolate from China, a set of four specific primers was designed based on the sequence. The MgCl2 concentration and the reaction temperature were optimized to 6 mM, 64°C, respectively. LAMP products were detected by visual inspection of a color change due to addition of SYBR Green I stain. The specificity and sensitivity of the LAMP assay were determined. No cross-reaction was observed with other fish DNA viruses including eel herpesvirus, koi herpesvirus, and Chinese giant salamander iridovirus. The LAMP assay was found to be equally sensitive as nested PCR. A comparative evaluation of 10 fish samples using LAMP and nested PCR assays showed an overall correlation in positive and negative results for CyHV-2. These results indicate that the LAMP assay is simple, sensitive, and specific and has a great potential use for CyHV-2 detection in the laboratory and field.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Fish Diseases / diagnosis*
  • Fish Diseases / genetics*
  • Fish Diseases / virology
  • Goldfish
  • Herpesviridae Infections / diagnosis*
  • Herpesviridae Infections / genetics*
  • Herpesvirus 2, Human / genetics*
  • Molecular Sequence Data
  • Nucleic Acid Amplification Techniques / methods*
  • Time Factors