Comparison of the efficiency and sensitivity of virus isolation and molecular methods for routine diagnosis of infectious haematopoietic necrosis virus and infectious pancreatic necrosis virus

Abstract

Infectious haematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are widely distributed fish pathogens in Europe. A reverse transcriptase–polymerase chain reaction (RT–PCR) assay was developed for the detection of both viruses as an alternative method to virus assay in cell culture. Oligonucleotide primers corresponding to highly conserved regions of glycoprotein G‐gene sequences were used for IHNV. For the detection of IPNV the VP2‐coding region was selected for RT–PCR amplification. Products of the expected size were amplified from total ribonucleic acid (RNA) extracts of infected cells. The optimized RT–PCR methods successfully detected viral RNA from ovarian and seminal fluids and other organs. To enhance the sensitivity and specificity of RT–PCR, a semi‐nested PCR assay was tested using additional specific inner primers for reamplification of products obtained by RT–PCR. Because of the possibility of template carry‐over contamination, a closed one step RT–PCR method was tested. This technically simplified approach was then combined with the PCR–enzyme linked immunosorbent assay (ELISA) method for the detection of amplification products and verification using specific biotinylated probes. The test provides an additional tool for the detection of IHNV and IPNV which is rapidly and easily performed and is highly sensitive, especially for the detection of IHNV in fish samples coinfected with IPNV. The PCR–ELISA method for the detection of RT–PCR products enables the screening of large numbers of samples and offers the possibility for automatisation of diagnostic work.