Validation of high throughput methods for tissue disruption and nucleic acid extraction for ranaviruses (family Iridoviridae )

Viruses in the family Iridoviridae cause severe disease and economic loss in a wide range of cultured, wild and ornamental fish species in many countries, justifying the development and validation of diagnostic tests. In an effort to improve the efficiency of test protocols, in this study manual and high throughput semi-automated methods for tissue homogenisation and nucleic acid purification were compared for the detection of Epizootic Haematopoeitic Necrosis Virus (EHNV). The effectiveness of these methods at releasing EHNV particles and obtaining DNA was evaluated by virus isolation in bluegill fry (BF-2) cells, enzyme-linked immunosorbent assay (ELISA) and quantitative polymerase chain reaction (qPCR). Samples were prepared using tissue from infected redfin perch (Perca fluviatilis). Antigen and DNA yield were greater after homogenisation by bead-beating than by manual grinding of tissues from experimentally infected fish, which contained low quantities of virus. Bead-beating was compatible with virus isolation. There was no difference between the manual and semi-automated methods using samples from naturally infected fish which contained large amounts of virus. There was no difference in DNA yield between manual and semi-automated nucleic acid extraction for experimentally infected fish, however nucleic acid yields were greater after manual extraction for samples from naturally infected fish. Semi-automated tissue homogenisation and nucleic acid extraction required the least amount of time and were the most cost effective. The results of this study can be used as a guide to the selection of sample preparation procedures for other ranaviruses and probably more widely.