DNA PROBES FOR DIAGNOSIS OF GONOCOCCAL DISEASE

一种高效、经济的诊断试验的开发 对于淋病奈瑟菌的感染是这个项目的目标。 虽然目前的诊断方法灵敏而特异， 它们既耗时又昂贵。因为巨大的 淋球菌感染在美国的发病率，潜在的 这种测试的商业应用非常好。这是第一阶段 一项提案审查了两个DNA“探针”的可行性 诊断试剂。一种是基于一种 奈瑟氏菌基因组中发现的26个碱基对重复元件 SPP.一种21个碱基的寡核苷酸将被商业化合成 酶促生物素化。第二个探测器将是单个探测器 淋球菌外膜DNA链状片段 蛋白III基因。这个探测器将被生物素化。一个 该基因的限制性内切酶片段显示最佳物种 特异性将通过杂交分析来确定 几种奈瑟氏菌的基因组DNA。这两个探测器都将是 用提纯的淋球菌DNA和 硝化棉的杂交分析。生物素化DNA探针 将用链霉亲和素和生物素标记的络合物进行检测 碱性磷酸酶或尿素酶。将考虑将探测器用于 进一步发展(第二阶段)，如果他们的敏感性水平允许的话 检出不到万株淋球菌，意义不大 背景“噪音”。

Development of an efficacious and cost-effective diagnostic test for Neisseria gonorrhoeae infection is the goal of this project. Although current diagnostic methods are sensitive and specific, they are time consuming and costly. Because of the enormous morbidity of gonococcal infection in the U.S., the potential for commercial application of such a test is excellent. This phase I proposal examines the feasibility of two DNA "probes" as diagnostic reagents. One will be based on the DNA sequence of a 26 base-pair repetitive element found in the genomes of Neisseria spp. A 21 base oligonucleotide will be commercially synthesized and enzymatically biotinylated. The second probe will be a single stranded DNA fragment of the gonococcal outer membrane protein III gene. This probe will be biotinylated chemically. A restriction fragment of this gene demonstrating the best species specificity will be determined by hybridization analysis with genomic DNA from several Neisseria spp. Both probes will be examined for sensitivity using purified gonococcal DNA and hybridization analysis on nitrocellulose. Biotinylated DNA probes will be detected with complexes of streptavidin and biotinylated alkaline phosphatase or urease. Probes will be considered for further development (Phase II), if their level of sensitivity allows detection of less than ten thousand gonococci with insignificant background "noise".