Analytical validation of one-step realtime RT-PCR for detection of infectious hematopoietic necrosis virus (IHNV)

Infectious hematopoietic necrosis virus (IHNV) is the causative agent of infectious hematopoietic necrosis, a disease listed by both the European Union (EU) and the World Organisation for Animal Health (OIE). Recently, molecular methods based on real-time RT-PCR have been approved for surveillance of IHNV within the EU, using the two-step real-time RT-PCR developed by Purcell et al. (2013). The two-step procedure has some disadvantages compared to one-step methods due to the higher risk of contamination and it being a more laborious test. Here we show that modifying the original protocol to one-step real-time RT-PCR does not significantly compromise the analytical parameters of the method and it provides a similar detection limit for detection of IHNV when compared with cell culture methods. The modified one-step protocol is robust and reproducible as shown by an inter-laboratory proficiency test carried out in eight laboratories in Europe.