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

Maja A,  Zaczek-Moczydlowska; Letitia Mohamed- Smith; Anna Toldra; Chantelle Hooper; Monica Campas; M. Dolors Furones; Tim Bean; Katrina Campbell

doi:10.3390/ijms21186605

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

Maja A, Zaczek-Moczydlowska, Letitia Mohamed- Smith, Anna Toldra, Chantelle Hooper, Monica Campas, M. Dolors Furones, Tim Bean, Katrina Campbell

Royal (Dick) School of Veterinary Studies

Research output: Contribution to journal › Article › peer-review

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 herpesvirus 1 (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 additional Tte UvrD helicase enzyme used in the reaction was also evaluated with an improved reaction time of 10 minutes. Additionally, this study provides a robust workflow for optimization of primers for uncultured viruses using designed target plasmid, when DNA availability is limited.

Original language	English
Journal	International Journal of Molecular Sciences
DOIs	https://doi.org/10.3390/ijms21186605
Publication status	Published - 9 Sept 2020

Keywords / Materials (for Non-textual outputs)

colorimetric
Ostreid herpesvirus 1 (OsHV-1
loop-mediated isothermal amplification (LAMP)
oyster
C. gigas
early warning detection

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10.3390/ijms21186605

Ijms 21 06605 v2
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title = "A Single-Tube HNB-Based Loop-Mediated Isothermal Amplification for the Robust Detection of the Ostreid Herpesvirus 1",

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 herpesvirus 1 (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 additional Tte UvrD helicase enzyme used in the reaction was also evaluated with an improved reaction time of 10 minutes. Additionally, this study provides a robust workflow for optimization of primers for uncultured viruses using designed target plasmid, when DNA availability is limited. ",

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language = "English",

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AU - Zaczek-Moczydlowska, Maja A,

AU - Mohamed- Smith, Letitia

AU - Toldra, Anna

AU - Hooper, Chantelle

AU - Campas, Monica

AU - Furones, M. Dolors

AU - Bean, Tim

AU - Campbell, Katrina

PY - 2020/9/9

Y1 - 2020/9/9

N2 - 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 herpesvirus 1 (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 additional Tte UvrD helicase enzyme used in the reaction was also evaluated with an improved reaction time of 10 minutes. Additionally, this study provides a robust workflow for optimization of primers for uncultured viruses using designed target plasmid, when DNA availability is limited.

AB - 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 herpesvirus 1 (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 additional Tte UvrD helicase enzyme used in the reaction was also evaluated with an improved reaction time of 10 minutes. Additionally, this study provides a robust workflow for optimization of primers for uncultured viruses using designed target plasmid, when DNA availability is limited.

KW - colorimetric

KW - Ostreid herpesvirus 1 (OsHV-1

KW - loop-mediated isothermal amplification (LAMP)

KW - oyster

KW - C. gigas

KW - early warning detection

U2 - 10.3390/ijms21186605

DO - 10.3390/ijms21186605

M3 - Article

SN - 1422-0067

JO - International Journal of Molecular Sciences

JF - International Journal of Molecular Sciences

ER -

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

Abstract

Keywords / Materials (for Non-textual outputs)

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