Development of a biotinylated DNA probe for detection and identification of infectious hematopoietic necrosis virus

• Published in: Diseases of aquatic organisms Vol. 11; no. 1; pp. 57 - 65
• Main Authors: DEERING, R. E, ARAKAWA, C. K, OSHIMA, K. H, O'HARA, P. J, LANDOLT, M. L, WINTON, J. R
• Format: Journal Article
• Language: English
• Published: Oldendorf Inter-Research 01.01.1991
• Subjects: Biological and medical sciences; Brackish; Freshwater; Fundamental and applied biological sciences. Psychology; infectious hematopoietic necrosis virus; Marine; Microbiology; Oncorhynchus; Oncorhynchus mykiss; Oncorhynchus nerka; Oncorhynchus tshawytscha; Virology; Infection; Virus; Infectious hematopoietic necrosis virus; RNA; Rhabdoviridae; DNA; Viral disease; Identification; Method; Detection
• ISSN: 0177-5103; 1616-1580
• DOI: 10.3354/dao011057

A nonradioactive DNA probe assay was developed to detect and identify infectious hematopoietic necrosis virus (IHNV) using a dot blot format, The probe, a synthetic DNA oligonucleotide labeled enzymatically with biotin, hybridized specifically with nucleocapsid mRNA extracted from infected cells early in the virus replication cycle. A rapid, guanidinium thiocyanate based, RNA extraction method using RNAzol B and microcentrifuge tubes efficiently produced high quality RNA from 3 commonly used fish cell lines, CHSE-241, CHH-1, and EPC. The probe reacted with 6 diverse isolates of IHNV, but did not react with 2 related rhabdoviruses of fish, viral hemorrhagic septicemia virus and Hirame rhabdovirus. The biotinylated probe was sensitive, detecting picogram levels of target mRNA. Detection and identification of IHNV required 2 d when cells were inoculated at multiplicities of infection (MOI) greater than 2. Five days were necessary to detect and identify IHNV in cells inoculated at a MOI of 0.0002.