Abstract
The diagnostic properties of the one-step real-time reverse-transcription polymerase chain reaction assay for viral haemorrhagic septicaemia virus detection were compared to methods currently in use in the Czech Republic, namely, virus isolation using the cell culture and conventional reverse-transcription polymerase chain reaction followed by the nested polymerase chain reaction. The assays were tested on a panel of 25 archived viral haemorrhagic septicaemia isolates and 8 archived infectious haematopoietic necrosis isolates obtained from monitoring and/or outbreaks of the diseases among farmed salmonids in the Czech Republic. The ability to detect the presence of the virus in the tissues of fish was tested on additional 32 field samples collected from the rainbow trout (Oncorhynchus mykiss), brown trout (Salmo trutta) and brook trout (Salvelinus fontinalis). The real-time assay showed the highest analytic sensitivity by detecting the presence of viral nucleic acid in samples with 10-7 dilution, whereas the sensitivity of the conventional polymerase chain reaction peaked at 10-5. Diagnostic specificity of both molecular assays was confirmed by absence of cross-reactivity with the infectious haematopoietic necrosis virus isolates. This, along with consistent results in the detection of the virus in the fish tissues, confirms that the one-step real-time reverse-transcription polymerase chain reaction is currently an optimal stand-alone diagnostic method for the detection of the viral haemorrhagic septicaemia virus.
Highlights
Viral haemorrhagic septicaemia virus (VHSV) from the family Rhabdoviridae is the most important viral pathogen of salmonids worldwide
The diagnostic properties of the one-step real-time reverse-transcription polymerase chain reaction assay for viral haemorrhagic septicaemia virus detection were compared to methods currently in use in the Czech Republic, namely, virus isolation using the cell culture and conventional reverse-transcription polymerase chain reaction followed by the nested polymerase chain reaction
Diagnostic sensitivity and specificity of the assay was compared to the diagnostic methods currently in use, namely isolation on the cell culture and conventional RT-PCR followed by nested PCR
Summary
Viral haemorrhagic septicaemia virus (VHSV) from the family Rhabdoviridae is the most important viral pathogen of salmonids worldwide. The enveloped single-stranded ribonucleic acid virus causes outbreaks of the viral haemorrhagic septicaemia (VHS) amongst over 80 fresh- and saltwater fish species and has an increasing economic impact due to clinical signs, high mortality from the disease and costly eradication measures (Skall et al 2005). The currently used methods meet these criteria, but their disadvantages are high time requirements (isolation on the cell culture) or higher risk of sample contamination (conventional reverse-transcription polymerase chain reaction [RT-PCR], enzyme-linked immunosorbent assay [ELISA]) (Mackay 2004). The aim of the present study was to test the real-time RT-PCR assay, as described by Jonstrup et al (2013), on a panel of archived Czech VHSV isolates and field samples. Diagnostic sensitivity and specificity of the assay was compared to the diagnostic methods currently in use, namely isolation on the cell culture and conventional RT-PCR followed by nested PCR
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