Item talk:Q149956

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

A nonrad~oact~ve DNA probe assay was developed to detect and ~dent~fy infect~ous hernatopoiet~c necrosls virus (IHNV) uslng a dot blot format The probe a synthet~c DNA oligonucleot~de labeled enzymatlcally w~th biotln hybnd~zed spec~f~cally w~th nucleocaps~d mRNA extracted from Infected cells early In the vlrus repl~cation cycle A rap~d guan~dln~um th~ocyanate based RNA extraction method uslng RNAzol B and rn~crocentrifuge tubes eff~c~ently pioduced h~gh qual~ty RNA from 3 commonly used f~sh cell llnes, CHSE-214, CHH-1, and EPC The probe reacted with 6 d~verse ~solates of IHNV, but d~d not react \nth 2 related rhabdovlruses of fish viral hemorrhagic septlcemla vlrus and H~rame rhabdovlrus The b~ot~nylated probe was sensltlve detect~ng plcogram levels of target mRNA Detect~on and ~dentif~cat~on of IHNV requ~red 2d when cells wele lnoculated at n~ultiplic~t~es of infect~on (MOI) greater than 2 Flve days were necessary to detect and identify IHNV In cells lnoculated at a MO1 of 0 0002