Identification of a double-stranded RNA virus by using polymerase chain reaction and magnetic separation of the synthesized DNA segments.
AUTOR(ES)
Rimstad, E
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=268161Documentos Relacionados
- Improved double-stranded DNA sequencing using the linear polymerase chain reaction.
- Capped and conserved terminal structures in human rotavirus genome double-stranded RNA segments.
- Unique double-stranded RNAs associated with the Trichomonas vaginalis virus are synthesized by viral RNA-dependent RNA polymerase.
- The double-stranded RNA genome segments of cytoplasmic polyhedrosis virus are independently transcribed.
- Identification of double-stranded RNA-binding domains in the interferon-induced double-stranded RNA-activated p68 kinase.