EXTREMELY SENSITIVE ASSAYS FOR HUMAN RETROVIRUSES

Project Details

Description

Extremely sensitive assays will be developed for the detection of
pathogenic human retroviruses. The assays will be designed for the
screening of donated blood and for the identification of asymptomatic
carriers, in order to prevent the spread of T-lymphotropic
leukemia/lymphoma and acquired immunodeficiency syndrome (AIDS). Novel
recombinant RNA molecules will be prepared. They will serve as specific
hybridization probes for retroviral sequences and will also serve as
exponentially amplifiable reporters. Incubation of the hybrids with Q
beta replicase will generate as many as one billion RNA copies of each
probe, enabling the measurement of the number of retroviral targets in
the sample. The assays will not require the fractionation of cells or
cellular components; hybridization will take place in solution; hybrids
will be separated from unhybridized probes on magnetic beads;
amplification will require only a single incubation at 37C; and the
entire assay will take less than three hours. Assay formats will be
developed that will enable the simultaneous detection of different
pathogenic retroviruses in the same sample. Although the use of exponentially amplifiable probes theoretically allows
the detection of as little as one target molecule, the actual sensitivity
of these assays will be determined by the number of non-specifically
bound probes that persist, despite extensive washing of the hybrids. In
order to eliminate, or markedly reduce, this source of background, the
probe molecules will be altered to contain a molecular switch. This is a
region of the molecule that undergoes a conformational change when the
probe hybridizes to its target. Non-specifically bound probes will not
undergo this change. An additional enzymatic step will then be added to
the assay that will obligately link the replication of RNA reporters to
the state of the molecular switch. The use of binary probes in assays will also be explored. Two different
single-stranded DNA probes will be prepared. Each will comprise a
different portion of a template for the transcription of replicatable RNA
reporters. The probes will not be able to function as a template by
themselves: they will first have to hybridize to adjacent regions of the
target sequence, where they will be joined together by incubation with
DNA ligase. Since DNA ligase only works with hybridized DNAs, the
generation of replicatable RNA reporters from these templates will be
dependent on the hybridization of the probes to the target.
StatusFinished
Effective start/end date8/1/895/31/05

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $571,272.00
  • National Institutes of Health: $560,002.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health

ASJC

  • Medicine(all)

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