DNA PROBES FOR DIAGNOSIS OF GONOCOCCAL DISEASE

开发一种有效且具有成本效益的诊断测试 淋病奈瑟氏菌感染是这个项目的目标。 虽然目前的诊断方法是敏感和特异的， 它们既费时又费钱。 由于巨大的 美国淋球菌感染的发病率，的潜力 这种测试的商业应用是极好的。 这项I期 一项提案审查了两个DNA“探针”作为 诊断试剂 其中一个将基于一个人的DNA序列， 奈瑟氏菌基因组中发现的26个碱基对重复序列 spp. 将商业合成21个碱基的寡核苷酸 并酶促生物素化。 第二个探测器将是一个 淋球菌外膜DNA片段 蛋白III基因。 该探针将被化学生物素化。 一 该基因的限制性片段证明了最好的物种 特异性将通过杂交分析测定， 来自几种奈瑟氏菌属的基因组DNA 两个探测器都将 使用纯化的淋球菌DNA检查敏感性， 硝化纤维素杂交分析 生物素化DNA探针 将用链霉亲和素和生物素化的复合物检测 碱性磷酸酶或尿素酶。 探针将被考虑用于 进一步开发（第二阶段），如果其敏感性水平允许 检测不到1万个淋球菌， 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".