Rapid capture and detection of ostreid herpesvirus-1 from Pacific oyster Crassostrea gigas and seawater using magnetic beads

Abstract

Ostreid herpesvirus-1 (OsHV-1) has been involved in mass mortality episodes of Pacific oysters Crassostrea gigas throughout the world, causing important economic losses to the aquaculture industry. In the present study, magnetic beads (MBs) coated with an anionic polymer were used to capture viable OsHV-1 from two types of naturally infected matrix: oyster homogenate and seawater. Adsorption of the virus on the MBs and characterisation of the MB-virus conjugates was demonstrated by real-time quantitative PCR (qPCR). To study the infective capacity of the captured virus, MB-virus conjugates were injected in the adductor muscle of naïve spat oysters, using oyster homogenate and seawater without MBs as positive controls, and bare MBs and sterile water as negative controls. Mortalities were induced after injection with MB-virus conjugates and in positive controls, whereas no mortalities were recorded in negative controls. Subsequent OsHV-1 DNA and RNA analysis of the oysters by qPCR and reverse transcription qPCR (RT-qPCR), respectively, confirmed that the virus was the responsible for the mortality event and the ability of the MBs to capture viable viral particles. The capture of viable OsHV-1 using MBs is a rapid and easy isolation method and a promising tool, combined with qPCR, to be applied to OsHV-1 detection in aquaculture facilities.

Optimisation of a PMAxx™-RT-qPCR Assay and the Preceding Extraction Method to Selectively Detect Infectious Murine Norovirus Particles in Mussels

“…To address this issue, a novel assay for the extraction of infectious MNV from bivalve mollusc matrices was developed; this method, based on magnetic beads coated with anionic polymer, permits extraction of intact virus particles, representing a significant advantage over current proteinase K-based extraction methods, which damage viral capsids, thus precluding downstream assays to determine viral infectivity. Anionic polymer-coated beads have previously been demonstrated to capture different enveloped and non-enveloped from a range of matrices, including cell culture media, PBS, water, oyster homogenate and seawater (Hatano et al 2010;Patramool et al 2013;Sakudo and Onodera 2012;Toldrà et al 2018). Here, we demonstrate the capacity of anionic beads to capture infectious or inactivated MNV in PBS and from mussel DTs.…”

Portable electroanalytical nucleic acid amplification tests using printed circuit boards and open-source electronics

“…Future work will include molecular identification of other relevant DNA biomarkers, as well as subsequent assay characterization and validation. Ongoing research should focus on three specific areas: (i) shortening of the incubation times of the sandwich hybridization assay as well as performing stability studies of the SAMs on PCBs to achieve ready-to-use platforms; 59 (ii) integration of RPA on chip by using heating, 60,61 by performing a solid-phase amplification, 62 and by using microfluidics on PCBs 60 to achieve a true lab-on-a-chip device; and (iii) inclusion of a sample preparation step on chip by exploiting magnetic beads-powered cell capture 63 and enzymatic cell lysis and DNA extraction steps using paper. 7 Our demonstration of SAM-based PCB sensors will likely be applicable to many other SAM-based sensors.…”

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

“…DNA extracts were tested for concentration and quality using a Nanodrop (Thermo Fisher Scientific, Waltham, MA, USA) or/and Agilent 2200 Tape Station and quantified with the method provided for the plasmid. For qPCR analysis, homogenate lysates were prepared with the method previously reported [ 38 ], were confirmed and were quantified using OsHVDPFor/OsHVDPRev primer set [ 39 ] using an ABI 7300 thermocycler (Thermo Fisher Scientific, Waltham, MA, USA) according to qPCR conditions reported previously [ 38 ].…”