Project Details


A method for the detection of solitary molecules of viral RNA and DNA in
crude biological samples will be developed. this proposal extends current
investigations that employ recombinant RNAs as both specific hybridization
probes and templates for exponential RNA synthesis by Qbeta replicase. The
method will utilize binary probes that are two different single-stranded
DNAs, each comprising a different portion of a template for the synthesis
of a replicatable RNA reporter, and a different segment of a probe
sequence. The probes will not be able to function as a template by
themselves, but will give rise to such a template upon hybridization to a
target sequence if a target sequence is present in the sample.
Hybridization will take place in a crude lysate, and the hybrids will be
separated from unbound probes and from cellular debris on magnetic beads.
the hybridized binary probes will then be converted by either ligation or
extension into a functional template, which will then be transcribed to
produce replicatable RNA. The RNA will be exponentially amplified by Qbeta
replicase. Amplification will occur in a thin layer of a solid medium,
where every RNA molecule will give rise to a separate "colony". Since RNAs
will include short "replicas" of the target molecule, they will be
identified by blotting onto a membrane, followed by hybridization with
labeled oligonucleotides. The assay format will be extended to
simultaneously detect different pathogenic viruses in the same sample.
Also, the possibility of utilizing a new efficient Qbeta replicase template
(RQ135 RNA) for the replicatable probes, in addition to the currently used
MDV-1 RNA, will be explored.
Effective start/end date8/10/928/9/93


  • National Institutes of Health


  • Medicine(all)

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