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.

Rapid Diagnostic Test to Detect and Discriminate Infectious Hematopoietic Necrosis Virus (IHNV) Genogroups U and M to Aid Management of Pacific Northwest Salmonid Populations

“…All three of these isolates were obtained from Idaho rainbow trout farms, an industry and region associated with unusually high rates of IHNV genetic diversity [ 32 , 33 ]. In Germany, reduced sensitivity has been reported for the N Uni RT-rPCR when detecting E genogroup isolates associated with the rainbow trout industry due to the presence of nucleotide substitutions in the probe region [ 34 ]. A redesigned probe, implemented as a one-step PCR protocol, showed improved sensitivity for a limited number of German E isolates of IHNV tested in the study.…”

A brief review of novel nucleic acid test biosensors and their application prospects for salmonids viral diseases detection

“…The viral culture needs too much time for isolating and authenticating. Moreover, there is no sensitive cell lines for some viruses of aquatic animals [2,4] . Enzyme-linked immunosorbent assay (ELISA) is the most typical immunological method, which could detect virus antigen or antibody.…”

“…The RT-LAMP methods were also successfully developed to detect the IPNV [20,34,37] . Rodriguez et al [4] evaluated the five serological methods and RT-PCR and found that RT-PCR were the most appropriate and sensitive methods for the routine detection of IPNV from affected fish. Therefore, the nucleic acid test is the most commonly used detection method in the diagnosis of aquaculture viral pathogens.…”

“…They have seriously affected the development of the farmed salmonids industry of China. It is significant that disease causing agents are rapidly identified and differentiated [4] . For this reason, early and accurate nucleic acid detection is crucial and essential in preventing and controlling the spread of salmonids diseases [5,6] .…”

Stability of viral haemorrhagic septicaemia virus, infectious hematopoietic necrosis virus and cyprinid herpesvirus 3 in various water samples

“…The RT-qPCR of IHNV and VHSV samples were performed using a RT-qPCR kit (iTaq universal probes one-step kit, Bio-Rad) according to Chico et al (2006) and Hoferer et al (2019), respectively, and the protocols recommended by the Bavarian State Office for Health and Food Safety (LGL). As the negative control nuclease-free water was used and extraction and amplification controls were prepared using intype IC-RNA (Indical Bioscience GmbH) and specific primers and probe (Sigma-Aldrich).…”