Polymerase Chain Amplification of Infectious Hematopoietic Necrosis Virus RNA Extracted from Fixed and Embedded Fish Tissue

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Abstract
Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus that infects salmonid fish and, as a result, causes great economic losses to fish hatcheries. The direct detection of IHNV RNA in infected tissue would be useful in diagnosing the disease and preventing its spread. In this report, we describe a sensitive method for detecting IHNV RNA in formalin-fixed, paraffinembedded tissues of rainbow trout and steelhead Oncorhynchus mykiss. The technique is capable of detecting viral RNA in samples that have remained at room temperature in 10% buffered formalin for over 2 years. The RNA extracted from the deparaffinized tissue sections by phenol and guanidinium thiocyanate was amplified with primers to the viral nucleocapsid (N) gene with a heatstable DNA polymerase capable of reading both DNA and RNA. Prolonged incubation of the firststrand complementary DNA synthesis reaction for 20 min at 70°C was optimal for the synthesis of an N-specific, 252-base-pair product.

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