Quantification of DNA using the luminescent oxygen channeling assay

Background: Simplified and cost-effective methods for the detection and qua ntification of nucleic acid targets are still a challenge in molecular diag nostics. Methods: Luminescent oxygen channeling assay (LOCI(TM)) latex particles can be conjugated to synthetic oligodeoxynucleotides and hybridized, via linki ng probes, to different DNA targets. These oligomer-conjugated LOCI particl es survive thermocycling in a PCR reaction and allow quantified detection o f DNA targets in both real-time and endpoint formats. The endpoint DNA quan tification format utilized two sensitizer bead types that are sensitive to separate illumination wavelengths. These two bead types were uniquely annea led to target or control amplicons, and separate illuminations generated ti me-resolved chemiluminescence, which distinguished the two amplicon types. Results: In the endpoint method, ratios of the two signals allowed determin ation of the target DNA concentration over a three-log range. The real-time format allowed quantification of the DNA target over a six-log range with a linear relationship between threshold cycle and log of the number of DNA targets. Conclusions: This is the first report of the use of an oligomer-labeled lat ex particle assay capable of producing DNA quantification and sequence-spec ific chemiluminescent signals in a homogeneous format. It is also the first report of the generation of two signals from a LOCI assay. The methods des cribed here have been shown to be easily adaptable to new DNA targets becau se of the generic nature of the oligomer-labeled LOCI particles. (C) 2000 A merican Association for Clinical Chemistry.