DEVELOPMENT OF A BIOTINYLATED DNA PROBE FOR DETECTION AND IDENTIFICATION OF INFECTIOUS HEMATOPOIETIC NECROSIS VIRUS

被引：13

作者：

DEERING, RE ^{[1
]}

ARAKAWA, CK ^{[1
]}

OSHIMA, KH ^{[1
]}

OHARA, PJ ^{[1
]}

LANDOLT, ML ^{[1
]}

WINTON, JR ^{[1
]}

机构：

[1] UNIV WASHINGTON,SCH FISHERIES,SEATTLE,WA 98195

来源：

DISEASES OF AQUATIC ORGANISMS | 1991年 / 11卷 / 01期

关键词：

D O I：

10.3354/dao011057

中图分类号：

S9 [水产、渔业];

学科分类号：

0908 ;

摘要：

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-214, 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.

引用

页码：57 / 65

页数：9