Bovine viral diarrhea virus: molecular cloning of genomic RNA and its diagnostic application
Molecular cloning of a field isolate of bovine viral diarrhea virus (BVDV) strain 72 RNA was done in this study. The sensitivity and specificity of cloned cDNA sequences in hybridization assays with various BVDV strains were determined. cDNA was synthesized from polyadenylated BVDV RNA templates with oligo-dT primers, reverse transcriptase, and DNA polymerase I. The newly synthesized double-stranded BVDV cDNA was C-tailed with terminal deoxytransferase and annealed into G-tailed, Pst-1-cut pUC9 plasmid. Escherichia coli was transformed with the recombinant plasmids and a library of approximately 200 BVDV specific cDNA clones varying in length from 0.5 to 2.6 kilobases were isolated. The sensitivity and specificity of hybridization between the labelled cDNA and BVDV target sequences were determined. Cloned BVDV sequences were isolated from pUC9 plasmid DNA and labelled with /sup 32/P by nick translation. The detection limit by dot blot hybridization assay was 20 pg of purified genomic BVDV RNA. cDNA hybridization probes were specific for all strains of BVDV tested, regardless of whether they were noncytopathic and cytopathic, but did not hybridize with heterologous bovine viruses tested. Probes did not hybridize with uninfected cell culture or cellular RNA. Hybridization probes were at least as sensitive as infectivity assays in detecting homologous virus.
- Research Organization:
- Tennessee Univ., Knoxville (USA)
- OSTI ID:
- 6257173
- Resource Relation:
- Other Information: Thesis (Ph. D.)
- Country of Publication:
- United States
- Language:
- English
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DNA
HYBRIDIZATION
RNA
DNA-CLONING
VIRUSES
AMINO ACID SEQUENCE
CATTLE
ESCHERICHIA COLI
PHOSPHORUS 32
PLASMIDS
TRACER TECHNIQUES
TRANSFERASES
ANIMALS
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CELL CONSTITUENTS
CLONING
DAYS LIVING RADIOISOTOPES
DNA HYBRIDIZATION
DOMESTIC ANIMALS
ENZYMES
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
MAMMALS
MICROORGANISMS
MOLECULAR STRUCTURE
NUCLEI
NUCLEIC ACIDS
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PARASITES
PHOSPHORUS ISOTOPES
RADIOISOTOPES
RUMINANTS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques