EVALUATION OF A NONRADIOACTIVE ASSAY FOR THE DETECTION OF AMPLIFIED DNA

这个项目的重点是评估一种使用聚合酶 链反应（PCR）扩增和时间分辨荧光法 （TRF）检测特定靶DNA。扶轮基金会使用长寿的 镧系元素离子如铕的荧光性质。在扶轮基金会， 用短波脉冲激发荧光镧系元素， 在背景荧光已经 腐烂了TRF已成功地应用于各种非同位素 免疫测定和DNA杂交测定。的一个主要 这种方法的优点是它是非放射性的， 适用于DNA探针分析的常规转移 临床实验室在该测定格式中，PCR通过标准品 程序，并且所需DNA产物的产生是 使用铕标记的寡核苷酸探针检测。约束 在时间分辨荧光计（Delfia Research）中测量铕 荧光计，Wallac，图尔库，芬兰）。的敏感性和 正在对测定的特异性进行评价， 微生物包括巨细胞病毒、分枝杆菌 结核病和微孢子虫物种。该试验正在进行中 与Southern印迹杂交比较。到目前为止， 似乎在定性上与Southern印迹一样敏感 这些生物的杂交。目前正在努力确定 该方法作为定量DNA的手段的灵敏度。

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.