DETECTION OF SOLITARY MOLECULES OF VIRAL NUCLEIC ACIDS

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.