RAPID MICROBIAL DIAGNOSIS BY NUCLEIC ACID AMPLIFICATION

通过核酸扩增进行快速微生物诊断

• 批准号: 3547712
• 负责人: DOUGLAS D RICHMAN
• 金额: $ 17.7万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 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/3547712
• 关键词: Adenoviridae; Betaherpesvirinae; Chlamydia trachomatis; Coronaviridae; Epstein Barr virus; Neisseria gonorrhoeae; Pneumovirus; Treponema pallidum; diagnosis design /evaluation; diagnosis quality /standard; genital herpes; herpes simplex virus 1; herpes simplex virus 2; human herpesvirus 6; human tissue; keratoconjunctivitis; microorganism classification; ocular herpes; parainfluenza virus type 1; parainfluenza virus type 2; polymerase chain reaction; rapid diagnosis; respiratory infections; respiratory virus; rhinovirus; sexually transmitted diseases; varicella zoster virus; virus classification

A number of factors, especially the prospect of antiviral chemotherapy, have made the need for rapid, sensitive, specific and practical assays for microbial diagnosis increasingly apparent. Progress has been limited in developing rapid viral diagnostic methods largely because of the limited amount of viral analyte molecules (antigen and nucleic acid) in clinical material. The conceptual breakthrough of amplifying an analyte molecule with the polymerase chain reaction has provided new promise for the development of needed assays. The principal investigator and his colleagues have been investigating gene amplification methods to detect viral nucleic acid in clinical material utilizing HIV- I as the prototype agent. Most recently they have been able to detect the sequential appearance of the 4 nucleotide mutations in the reverse transcriptase gene that account for AZT resistance in serial lymphocyte samples from patients receiving AZT therapy. An alternative method for sequence amplification has been developed that in an isothermal (37 degrees C), single cycle reaction that can generate 10(6) copies in 45 minutes. A bead based sandwich capture assay has also been developed that permits reproducible detection and quantitation of the products of this new amplification scheme. This assay is also amenable to nonisotopic detection. A device and method for the simultaneous assay for multiple agents in a single specimen has also been developed. In this proposal methods will be optimized and then applied to four sets of target organisms:herpesviruses, respiratory viruses, genital pathogens and ophthalmologic pathogens. The principal investigator has extensive experience in the clinical diagnosis of antibodies, antigens, and nucleic acids. Large inventories of well characterized clinical specimens for each of the relevant agents are available for these investigations. A systematic, sequential approach will be pursued to fulfill the following aims: AIM 1: Identify nucleic acid sequences from target organisms that will provide organism specificity while maintaining broad reactivity with all strains of that organism. AIM 2: Apply the specific amplification and detection methods to clinical materials known to contain the target agent (and controls) to verify sensitivity, specificity, reproduceability and when possible quantitation. AIM 3: Once assays for individual agents in a panel for the sets of target organisms have been developed, apply the plastic chamber or derivative technologies to develop a rapid diagnostic method for a clinical syndrome (respiratory infection, genital lesions, keratoconjunctivitis).

多种因素，尤其是抗病毒化疗的前景， 需要快速、灵敏、特异和实用的检测方法 微生物诊断日益明显。进展有限 开发快速病毒诊断方法主要是因为有限 临床中病毒分析物分子（抗原和核酸）的量 材料。放大分析物分子的概念突破 聚合酶链式反应为 开发所需的检测方法。 首席研究员和他的同事一直在研究基因 检测临床材料中病毒核酸的扩增方法 使用HIV-I作为原型剂。最近他们已经能够 检测 4 个核苷酸突变的顺序出现 导致连续 AZT 耐药的逆转录酶基因 接受 AZT 治疗的患者的淋巴细胞样本。另一种选择 已开发出在等温条件下进行序列扩增的方法 (37℃)，单循环反应可在 45 时间内生成 10(6) 个拷贝 分钟。还开发了一种基于珠子的夹心捕获测定法 允许对这种新产品进行可重复的检测和定量 放大方案。该测定也适用于非同位素检测。 一种同时测定多种试剂的装置和方法 还开发了单一样本。 在本提案中，方法将被优化，然后应用于四组 目标微生物：疱疹病毒、呼吸道病毒、生殖器病原体和 眼科病原体。主要研究者拥有广泛的 具有抗体、抗原、核酸的临床诊断经验 酸。大量库存的特征明确的临床标本 相关代理人可参与这些调查。一个 将采取系统、连续的方法来实现以下目标 目标： 目标 1：鉴定目标生物体的核酸序列， 提供生物体特异性，同时保持与所有生物体的广泛反应性 该生物体的菌株。 目标2：将特异性扩增和检测方法应用于临床 已知含有目标物质（和控制物）的材料需要验证 灵敏度、特异性、再现性以及可能的定量。 目标 3：对一组靶标中的单个试剂进行一次分析 生物体已开发，应用塑料室或衍生物 开发临床综合征快速诊断方法的技术 （呼吸道感染、生殖器病变、角结膜炎）。