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. 2020 Sep 9;21(18):6605.
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-Moczydłowska  1 , Letitia Mohamed-Smith  1 , Anna Toldrà  2 , Chantelle Hooper  3 , Mònica Campàs  2 , M Dolors Furones  2 , Tim P Bean  4 , Katrina Campbell  1
Affiliations

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

Maja A Zaczek-Moczydłowska et al. Int J Mol Sci. .

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.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Nucleic acid sequence of the target OsHV-1 DNA fragment (235 bp) and designed LAMP primers binding sites; outer primers: F3 and B3 (red), inner primers: F2 and F1c (FIP) (orange), and B1c and B2 (BIP) (green) visualized using SnapGene Viewer® 5.0.4.
Figure 2
Figure 2
Maps showing the composition of two synthesized plasmids and primers binding to the target visualized using SnapGene 5.0.3.: (A) AbHV (3263 bp) and (B) OsHV-1 (3127 bp).
Figure 3
Figure 3
Amplification of OsHV-1 by LAMP assay: (A) electropherogram profile comparison of NTC (blue) and positive sample (OsHV-1 DNA-plasmid) (orange) analysed using Tape Station Analysis Software A.02.02 (Agilent Technologies, Cheshire, UK) with a progressive increase of the amplified LAMP product showing a size shift of 90–1000 bp; (B) gel image profile comparison: (L1)—ladder 15–5000 bp, (A1)—NTC and (B1)—positive samples showing a single high molecular band smear-like pattern (90 bp–1000 bp) of the amplified LAMP product visualized using Tape Station Analysis Software A.02.02 (Agilent Technologies, Cheshire, UK); and (C) NTC (violet) and positive samples (OsHV-1 DNA-plasmid) (blue) visualized using HNB.
Figure 4
Figure 4
LAMP primer evaluation for specific target amplification and applicability to detect OsHV-1 present in the sample through comparison of positive and negative templates: A1—L_1 (103 copies virus/reaction), B1—OsHV-1 plasmid (105 copies virus/reaction), C1—L_3 (103 copies virus/reaction), A2—NTC, B2—nuclease-free water (negative control), C2—UN_1 containing other DNA than the target (negative control) and D2—AbHV plasmid (negative control). (A) Electropherogram comparison profiles of positive and negative templates analysed using Tape Station Analysis Software A.02.02 (Agilent Technologies) for assessment of progressive increase of amplified LAMP product, with positive templates showing a size shift of 90–1000 bp; (B) gel image profile comparison of positive (A1, B1 and C1) and negative templates (A2, B2, C2 and D2) analysed using Tape Station Analysis Software A.02.02 (Agilent Technologies) for assessment of smear-like pattern bands; and (C) 6 colorimetric assessments of amplified LAMP products for positive samples (A1, B1 and C1) (light blue) and negative samples (A2, B2, C2 and D2) (violet) using HNB.
Figure 5
Figure 5
Electropherograms of NTC and five OsHV-1 plasmid standard amplifications in LAMP assay: (A) NTC—0 copies virus/reaction, (B) 101 copies virus/reaction, (C) 102 copies virus/reaction, (D) 103 copies virus/reaction, (E) 104 copies virus/reaction and (F) 105 copies virus/reaction. Colorimetric assessment of amplified LAMP products using HNB: (A1) NTC—0 copies virus/reaction, (B1) 101 copies virus/reaction, (C1) 102 copies virus/reaction, (D1) 103 copies virus/reaction, (E1) 104 copies virus/reaction and (F1) 105 copies virus/reaction.
Figure 6
Figure 6
LAMP primer evaluation for amplification of the OsHV-1 plasmid (T = 65 °C, t = 10 min) and sensitivity with the addition of Tte UvrD helicase enzyme: (A) electropherogram comparison profiles of positive (OsHV-1 plasmid) and negative templates (NTC, UN_1 and AbHV) analysed using Tape Station Analysis Software A.02.02 (Agilent Technologies) for assessment of progressive increase of amplified LAMP product. Positive templates show a size shift of 350–5000 bp. (B) Colorimetric assessment of amplified LAMP products for positive (OsHV-1) (pink) and NTC (violet) templates using HNB.
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