NOVEL ULTRASENSITIVE COLORIMETRIC ASSAY FOR DNA

特异DNA序列的检测在遗传诊断中的应用 疾病、癌症和传染病变得越来越多 很普遍。已经采用了许多创新的技术， 包括寡核苷酸连接试验和反向斑点杂交 杂交。终点，以及在一定程度上的有效性 这些检测中的一种是由DNA检测的灵敏度决定的 使用的技术。进展通过以下方式得到显著加强 聚合酶链式反应(PCR)的应用。然而，实施 聚合酶链式反应可能很昂贵，而且设备和专业知识密集。我们 已经应用了一种被证明有效的技术在 DNA的非同位素检测的免疫测定法.初始数据 表示敏感度提高10(2)-10(3)倍 传统的技术。这种我们称之为Edna-ELCA的化验方法使用 凝固级联反应的放大以产生 可用比色法检测的最终产品。DNA模板被标记为 生物素和半胱氨酸，然后固定在包被的链霉亲和素中 微量滴定板。针对半抗原的抗体与一种 蛇毒凝血激活剂(RVVXa)，并引入到 微量滴度很好。然后，通过添加 凝血因子，包括一种标记纤维蛋白原的酶。这个 标记纤维蛋白的酶的沉积是阳性的指示 反应。在本提案中，我们选择了三个具体的应用 以证明该方法的实用性。它们是对DeltaF508的检测 囊性纤维化突变，K-1基因第一外显子第12位密码子突变 RAS在结肠癌中的作用，以及甲氧西林耐药中的mecA基因 金黄色葡萄球菌。

The detection of specific DNA sequences for the diagnosis of genetic diseases, cancer, and infectious disease is becoming increasingly widespread. A number of innovative techniques have been employed, including the oligonucleotide ligation assay and reverse dot-blot hybridization. The end-point, and to a certain extent the effectiveness of these assays is determined by the sensitivity of the DNA detection techniques used. Progress has been significantly enhanced through the use of the polymerase chain reaction (PCR). However, implementation of PCR can be expensive, as well as equipment and expertise intensive. We have applied a technique with proven effectiveness in the field of immunoassays for the non-isotopic detection of DNA; initial data indicates an increase in sensitivity 10(2)-10(3) times higher than conventional techniques. The assay, which we have called EDNA-ELCA, uses the amplification of the coagulation cascade to produce a colorimetrically detectable end product. A DNA template is labelled with biotin and a haptene, and is then immobilised in a streptavidin coated microtiter plate. An antibody against the haptene is conjugated to a snake venom coagulation activator (RVVXa), and introduced into the microtiter well. The assay is then developed by the addition of coagulation factors, including an enzyme labelled fibrinogen. The deposition of enzyme labelled fibrin is indicative of a positive reaction. In this proposal we have chosen three specific applications to prove the utility of this method. They are detection of the deltaF508 mutation of cystic fibrosis, a codon 12 mutation in the first exon of K- ras for colon cancer, and the mecA gene in methicillin resistant Staphlococcus aureus.