Development of a biotinylated DNA probe for detection and identification of infectious hematopoietic necrosis virus
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 ear...
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Published in | Diseases of aquatic organisms Vol. 11; no. 1; pp. 57 - 65 |
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Main Authors | , , , , , |
Format | Journal Article |
Language | English |
Published |
Oldendorf
Inter-Research
01.01.1991
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Subjects | |
Online Access | Get full text |
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Summary: | 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. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0177-5103 1616-1580 |
DOI: | 10.3354/dao011057 |