Improvement of a diagnostic procedure in surveillance of the listed fish diseases IHN and VHS

Abstract

Infectious haematopoietic necrosis (IHN) and viral haemorrhagic septicaemia (VHS) are OIE-listed and notifiable viral fish diseases which are controlled by eradication and surveillance programmes globally. The present study provides improved RT-qPCR procedures based on recently described OIE protocols. Improvements comprise the design of a new TaqMan® probe, replacing a TaqMan® MGB probe that turned out to show impaired binding. Reason for this is SNPs detected in the nucleoprotein N gene sequences of IHNV strains targeted by the RT-qPCR. Furthermore, the IHNV and VHSV RT-qPCR assays were realized as one-step and one-run procedures supplemented by an endogenous control system. The IHNV and VHSV RT-qPCR assays are characterized by a technical sensitivity of 19 and 190 gene equivalents (cRNA) and an analytical sensitivity of 2-7 and 13 TCID₅₀ /ml, respectively. For verification purposes, 105 IHNV and 165 VHSV isolates and several non-targeted viral and bacterial pathogens were included and returned adequate results. However, in field samples divergent results left 14 samples of 154 undetected for IHNV and one sample of 127 for VHSV using cell culture. The study shows that RT-qPCR assays ensure facilitated and reliable testing on IHNV and VHSV in eradication and surveillance programmes.

Keywords: RT-qPCR; diagnostic validation; endogenous control system; field validation; infectious haematopoietic necrosis virus; viral haemorrhagic septicaemia virus.