Identification of a double-stranded RNA virus by using polymerase chain reaction and magnetic separation of the synthesized DNA segments.
A double-nested polymerase chain reaction assay (PCR), followed by magnetic separation of the PCR-synthesized DNA segments, was developed to detect a double-stranded RNA virus, infectious pancreatic necrosis virus from salmonid fish. Viral RNA was extracted from cell cultures and used for cDNA synthesis. The cDNA produced was used as a template in a double PCR. The sensitivity of this double PCR was approximately 0.8 pg of template double-stranded RNA. The DNA segment produced from the first PCR was also used as a template in a second PCR with a set of two 5'-labeled primers, one with biotin and the other with 32P. The PCR segment that was then synthesized was separated from the solution by using streptavidin-coated, superparamagnetic beads. The levels of radioactivity measured in the magnetically separated fractions were significantly higher in the positive samples than they were in the negative samples.
Rimstad
E
E
Department of Microbiology and Immunology, Norwegian College of Veterinary Medicine.
Hornes
E
E
Olsvik
O
O
Hyllseth
B
B
D000818:Animals; D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D008280:Magnetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012328:RNA Viruses / Q000235:genetics / Q000302:isolation & purification*; D012330:RNA, Double-Stranded / Q000235:genetics / Q000302:isolation & purification; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D012487:Salmonidae / Q000382:microbiology
D004279:DNA, Viral; D012330:RNA, Double-Stranded; D012367:RNA, Viral