AMPLIFIED NUCLEIC ACID-EIA FOR MICROBIAL DIAGNOSIS

用于微生物诊断的扩增核酸-EIA

• 批准号: 3547663
• 负责人: ROBERT H YOLKEN
• 金额: $ 21.61万
• 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/3547663
• 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; 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于管理具有重要意义 以及控制疾病爆发。 最 先前开发的测定系统已经利用免疫测定或 用于检测微生物的核酸杂交程序。 免疫测定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， 检测所有微生物。 我们建议将联合收割机固相 捕获、核酸扩增和免疫测定方法， 开发用于检测微生物病原体的单一测定系统。 靶核酸将通过以下方式特异性结合到固相表面： 与RNA探针杂交。 在去除污染后， 核酸和其它材料，结合的核酸将 通过循环酶促反应洗脱和扩增。 的 扩增的核酸然后通过固相定量 酶免疫测定程序。 该测定格式将允许 特异性微生物基因组的灵敏扩增和检测 人体液中的序列，而不受外来干扰 材料. 此外，利用常见的酶免疫分析 反应定量的程序将允许利用 广泛应用于临床和研究实验室的分析系统 环境. 将开发捕获扩增试验，用于检测 肠道、呼吸道和性传播的病原体 疾病 最初的靶微生物包括轮状病毒、流感病毒 病毒和沙眼衣原体。 灵敏度和特异性试验 将使用纯化的核酸和 同源和异源微生物的范围。 临床 然后将评价测定系统的效率和特异性 利用从明确定义的研究人群中获得的系列样品。 的 这些检测方法的成功开发之后， 其他微生物剂的类似检测。 生产 这些测定的应用可能会改善患者的医疗护理， 疑似感染和尽量减少疾病在医院内传播 环境.