Rotavirus gene detection with biotinylated single-stranded RNA probes.
Biotinylated single-stranded RNA probes from two of the eleven genome segments of the simian rotavirus SA11 were synthesized from cloned DNA and used in dot-blot and Northern-blot hybridization assays. Different types of membranes and conditions to prepare and use synthetic non-radioactive transcript probes were evaluated to obtain optimal test results. Nytran membranes showed the highest sensitivity and lowest backgrounds for hybridization with biotinylated RNA probes. When a gene 6 single-stranded biotinylated probe was used in a dot-blot format, test sensitivity was 0.1 ng for detection of homologous RNA and 0.4-1.5 micrograms for detection of RNA from heterologous rotavirus strains. When used in Northern blots, detection with this gene 6 probe required 1 ng of total SA11RNA or 50 ng of heterologous RNA to be applied to the gels for transfer. Simultaneous hybridization with probes from two different genes on one membrane showed a detection level similar to that seen with single probes alone. The advantages of using biotinylated single-stranded RNA probes to detect or characterize the genes of viruses with double-stranded RNA genomes are shown.
Bellinzoni
R
R
Division of Molecular Virology, Baylor College of Medicine, Houston, TX 77030.
Xi
J A
JA
Tanaka
T N
TN
Scodeller
E
E
Estes
M K
MK
D001710:Biotin; D015152:Blotting, Northern / Q000379:methods; D005814:Genes, Viral; D015151:Immunoblotting / Q000379:methods; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D015347:RNA Probes; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D012401:Rotavirus / Q000032:analysis* / Q000235:genetics; D014158:Transcription, Genetic
D015347:RNA Probes; D012367:RNA, Viral; D001710:Biotin