A Single-Tube HNB-Based Loop-Mediated Isothermal Amplification for the Robust Detection of the Ostreid herpesvirus 1

Int J Mol Sci. 2020 Sep 9;21(18):6605. doi: 10.3390/ijms21186605.

Abstract

The Ostreid herpesvirus 1 species affects shellfish, contributing significantly to high economic losses during production. To counteract the threat related to mortality, there is a need for the development of novel point-of-care testing (POCT) that can be implemented in aquaculture production to prevent disease outbreaks. In this study, a simple, rapid and specific colorimetric loop-mediated isothermal amplification (LAMP) assay has been developed for the detection of Ostreid herpesvirus1 (OsHV-1) and its variants infecting Crassostrea gigas (C. gigas). The LAMP assay has been optimized to use hydroxynaphthol blue (HNB) for visual colorimetric distinction of positive and negative templates. The effect of an additional Tte UvrD helicase enzyme used in the reaction was also evaluated with an improved reaction time of 10 min. Additionally, this study provides a robust workflow for optimization of primers for uncultured viruses using designed target plasmid when DNA availability is limited.

Keywords: C. gigas; LAMP; OsHV-1; POCT; colorimetric; early warning detection; oyster.

Publication types

  • Evaluation Study

MeSH terms

  • Animals
  • Crassostrea / virology
  • DNA Helicases
  • DNA Viruses / isolation & purification*
  • Molecular Diagnostic Techniques / methods*
  • Naphthalenesulfonates
  • Nucleic Acid Amplification Techniques / methods*

Substances

  • Naphthalenesulfonates
  • trisodium 3-hydroxy-4-((2Z)-2-(2-oxo-4-sulfonatonaphthalen-1-ylidene)hydrazinyl)naphthalene-2,7-disulfonate
  • DNA Helicases

Supplementary concepts

  • LAMP assay
  • Ostreid herpesvirus 1