AMPLIFIED NUCLEIC ACID-EIA FOR MICROBIAL DIAGNOSIS

用于微生物诊断的扩增核酸-EIA

• 批准号: 3547660
• 负责人: ROBERT H YOLKEN
• 金额: $ 19.29万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3547660
• 关键词: Chlamydia trachomatis; Rotavirus; communicable disease diagnosis; diagnosis design /evaluation; enzyme linked immunosorbent assay; gastrointestinal disorder diagnosis; human subject; microorganism classification; microorganism genetics; natural gene amplification; nucleic acid hybridization; nucleic acid probes; polymerase chain reaction; respiratory disorder diagnosis; respiratory infections; sexually transmitted diseases; virus classification; virus genetics

The rapid diagnosis of infectious diseases is important for the management of ill patients and for the containment of disease outbreaks. Most previously developed assay systems have made use of either immunoassay or nucleic acid hybridization procedures for the detection of micro-organisms. Immunoassay and hybridization techniques offer advantages for use in microbial diagnosis; however no single method has proven to be ideal for the detection of all microbial agents. We propose to combine solid phase capture, nucleic acid amplification, and immunoassay methodologies to develop a single assay system for the detection of microbial pathogens. Target nucleic acids will be specifically bound to solid phase surfaces by hybridization to RNA probes. Following the removal of contaminating nucleic acids and other materials by washing, the bound nucleic acids will be eluted and amplified by means of cyclical enzymatic reactions. The amplified nucleic acids will then be quantitated by means of solid phase enzyme immunoassay procedures. This assay format will allow for the sensitive amplification and detection of specific microbial genomic sequences in human body fluids without interference from extraneous materials. Furthermore, the utilization of familiar enzyme immunoassay procedures for reaction quantitation will allow for the utilization of the assay system in a wide range of clinical and research laboratory environments. The capture amplification assays will be developed for the detection of pathogenic agents of enteric, respiratory, and sexually transmitted diseases. Initial target organisms will include rotaviruses, influenza viruses and Chlamydia trachomatis. The sensitivity and specificity assays will be evaluated in a step-wise fashion using purified nucleic acids and a range of homologous and heterologous microorganisms. The clinical efficiency and specificity of the assay systems will then be evaluated utilizing serial samples obtained from well-defined study populations. The successful development of these assays will be followed by the development of analogous assays for additional microbial agents. The productive application of these assays might improve the medical care of patients with suspected infections and minimize disease transmission within hospital environments.

传染病的快速诊断对管理具有重要意义 用于治疗病人和控制疾病暴发。多数 以前开发的检测系统已经利用免疫检测或 用于检测微生物的核酸杂交程序。 免疫分析和杂交技术提供了用于 微生物诊断；然而，没有一种方法被证明是理想的 所有微生物制剂的检测。我们建议将固相结合 捕获、核酸扩增和免疫分析方法 开发微生物病原体检测的单一检测系统。 靶标核酸将通过以下方式与固相表面特异结合 与RNA探针杂交。在去除污染之后 通过洗涤核酸等物质，将结合的核酸 通过循环酶反应进行洗脱和扩增。这个 然后用固相法定量扩增的核酸。 酶免疫分析程序。这种化验格式将允许 特定微生物基因组的灵敏扩增和检测 不受外界干扰的人体体液中的序列 材料。此外，常见的酶免疫分析方法的应用 反应定量的程序将允许利用 广泛应用于临床和科研实验室的化验系统 环境。 将开发捕获扩增分析方法来检测 肠道、呼吸道和性传播的病原体 疾病。最初的目标生物将包括轮状病毒、流感 病毒和沙眼衣原体。灵敏度和特异度分析 将使用纯化的核酸和一个 同源和异源微生物的范围。临床部 然后将对检测系统的效率和特异性进行评估 利用从明确定义的研究总体中获得的系列样本。这个 在成功开发这些检测方法之后，将进行开发 其他微生物制剂的类似化验。多产的 应用这些检测方法可能会改善患者的医疗保健 疑似感染，尽量减少疾病在医院内的传播 环境。