EVALUATION OF A NONRADIOACTIVE ASSAY FOR THE DETECTION OF AMPLIFIED DNA

用于检测扩增 DNA 的非放射性测定法的评估

• 批准号: 2456713
• 负责人: E O'SHAUGHNESSY
• 金额: --
• 依托单位: CLINICAL CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2456713
• 关键词: DNA; Microsporidia; Mycobacterium tuberculosis; cytomegalovirus; evaluation /testing; fluorimetry; microorganism genetics; nucleic acid probes; oligonucleotides; polymerase chain reaction; southern blotting

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.

这个项目的重点是评估一种使用聚合酶的分析方法。 链式反应扩增和时间分辨荧光法 (TRF)检测特定的靶DNA。扶轮基金会使用长寿的 稀土离子，如Eu的荧光性质。在扶轮基金会， 用短波脉冲激发荧光镧系元素， 发射的光是在背景荧光 腐烂了。TRF已成功地应用于各种非同位素 免疫测定法和DNA杂交测定法。其中一个主要的 这种方法的优点是它是非放射性的，因此比 适用于将DNA探针分析转换为常规分析 临床实验室。在这种检测格式中，聚合酶链式反应是按标准进行的 程序，并且所需DNA产物的生产是 使用标记Eu的寡核苷酸探针进行检测。定界 在时间分辨荧光仪中测量Eu(Delfia Research 荧光计，沃尔拉克，图尔库，芬兰)。敏感度和 目前正在对多种检测方法的特异性进行评估 微生物包括巨细胞病毒、分枝杆菌 结核病和微孢子虫物种。化验正在进行中 与Southern印迹杂交结果进行比较。到目前为止，化验结果 在性质上似乎和Southern印迹一样敏感 为这些有机体进行杂交。目前正在进行确定 该方法作为DNA定量手段的灵敏度。