EVALUATION OF A NONRADIOACTIVE ASSAY FOR THE DETECTION OF AMPLIFIED DNA

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This project focuses on evaluating an assay that uses polymerase chain reaction (PCR) amplification and time-resolved fluorometry (TRF)to detect specific target DNA. TRF uses the long-lived fluorescence properties of lanthanide ions such as europium. In TRF, a fluorescent lanthanide is excited with a short-wave pulse, and the emitted light is measured after the background fluorescence has decayed. TRF has been successfully applied to various nonisotopic immunoassays and DNA hybridization assays. One of the main advantages of this method is that it is nonradioactive and thus more suited for the transfer of DNA probe analysis to the routine clinical laboratory. In this assay format the PCR is run by standard procedures, and the production of the desired DNA product is detected using a europium-labeled oligonucleotide probe. Bound europium is measured in a time-resolved fluorometer (Delfia Research Fluorometer, Wallac, Turku, Finland). The sensitivity and specificity of the assay are being evaluated for a number of microorganisms including cytomegalovirus, mycobacterium tuberculosis, and microsporidium species. The assay is being compared with hybridization by Southern blot. Thus far, the assay appears to be qualitatively as sensitive as Southern blot hybridization for these organisms. Work is ongoing to determine the sensitivity of this method as a means of quantifying DNA.