Development of a new diagnostic tool (Mini-Array) for ostreid Herpesvrus 1 (OsHV-1) detection in the Pacific oyster Crassostre gigas

• Main Authors: Varsamos, S., Pepin, Jean-francois, Sauvage, Christopher, Renault, Tristan
• Format: Conference Object
• Language: English
• Published: 13th EAFP International Conference on 'Diseases of Fish and Shellfish' 2007
• Subjects: Diagnostic; Crassotrea gigas; Oyster; Detection; Herpesviridae; OsHV 1; Pacific; Crassostrea gigas; Pacific oyster
• Online Access: https://archimer.ifremer.fr/doc/2007/acte-3652.pdf; https://archimer.ifremer.fr/doc/00000/3652/

OsHV-1 was the first herpesvirus to be identified in an invertebrate host and is associated to mortalities in the Pacific oyster (Crassostrea gigas) and other economically important bivalve species. Infections due to OsHV-1 are frequently reported and cause high mortality rates in larvae and spat of C. gigas. Detection of this pathogen is usually performed by PCR and/or in situ hybridization. This presentation will focus on the development of a new molecular diagnostic tool for OsHV-1 (and OsHV-1 variant 1) detection: the mini-array. This detection system, which allows multiple pathogens detection, combines viral DNA amplification by PCR and a specific hybridisation on a nylon membrane, revealed by a colorimetric reaction (DNA chip). Multiplex PCR involves specific primers amplifying sequences from OsHV-1. Hybridisation involves probes designed to specifically match to the PCR products. Detection of the obtained hybrids is performed using enzyme-linked antibodies producing a dark blue precipitate following addition of an appropriate substrate. Interpretation of the results is performed by the naked eye. Mini-array's main features include: double specificity, high sensitivity and standardisation of the process and reagents. Moreover, internal controls ensure limited risk of false positives & negatives samples. Subsequently, PCR, qPCR and mini-array methods have been submitted to a validation protocol (based on OIE recommendations and experts' advices) including rough estimation of analytical sensitivity and specificity, as well as repeatability. Two oyster populations with different prevalence of infection by OsHV-1 (high and low prevalence) have been tested to obtain rough estimations of diagnostic sensitivity & specificity. Inter-laboratory tests with reference samples should be the following step to confirm validation of these methods.