Validation of real time RT-PCR applied to cell culture for diagnosis of any known genotype of viral haemorrhagic septicaemia virus

Viral haemorrhagic septicaemia virus (VHSV), a member of the Rhabdoviridae family, is a major viral pathogen of cultured salmonid fish, and also infects a wide range of marine fish species. In the present study, two real time PCR protocols (based on SYBR Green and TaqMan (R)) were developed for the detection of strains belonging to all known genotypes of VHSV. Validation of the procedure, in terms of sensitivity, specificity and repeatability/reproducibility (R&R), was also performed. For this purpose, several pairs of primer amplifying regions corresponding to viral G and N genes were assayed. In the SYBR Green-based real time PCR, these primers failed to detect strains from some of the genotypes and/or showed low R&R. In order to improve the detection capacity, a multiplex procedure was designed, which enabled detection of all strains, with high R&R. The sensitivity of the procedure was measured, and a detection limit of 1 fg/mu l of viral RNA or 10 copies of cloned plasmid was established. On the other hand, the TaqMan probe-based multiplex real time PCR detected all European strains, with similar levels of sensitivity and R&R, but failed to detect the American types. (C) 2009 Elsevier B.V. All rights reserved.