Development and Assessment of a Sensitive and Cost-Effective Polymerase Chain Reaction to Detect Ostreid Herpesvirus 1 and Variants

Abstract

Ostreid herpesvirus 1 (OsHV-1) and variants (OsHV-1 var, OsHV-1 µvar, Irish genotype) have had a significant impact on the Pacific oyster, Crassostrea gigas, industry worldwide and on the survival of larvae and juveniles of several other bivalve species in Europe. Diagnostic techniques used to screen for this pathogen include histology, polymerase chain reaction (PCR), and quantitative polymerase chain reaction (qPCR), which allows for quantification of the virus. Techniques used to confirm infection include PCR, in situ hybridization, and transmission electron microscopy, which have variable sensitivity and specificity. In this study, several new primers were designed to amplify the C region in the ORF4 gene of the virus. This region is variable and diagnostic among OsHV-1 and variants. To date, the routinely used C2/C6 primers, which are used to screen for OsHV-1 and variants, amplify a product of 709 bp whereas the new primers described in this study amplify products of 296 bp, 385 bp, and 400 bp. Several C. gigas samples, which had been exposed to herpes virus and variants were screened, and a sample of wild mussels (Mytilus spp.) was used as a negative control. The performance of the new PCR and primers was compared with the performance of the C2/C6 primers and qPCR. The effect of tissue storage and DNA extraction on PCR performance was also examined. The results of this study indicate that the new PCR and primers are more rapid, sensitive, and cost-effective compared with the C2/C6 PCR, and are as sensitive as qPCR. The results also highlight the impact that sample storage, tissue selection, DNA extraction method used, and subsequent storage of DNA have on PCR success or failure.