RAPID MICROBIAL DIAGNOSIS BY NUCLEIC ACID AMPLIFICATION

通过核酸扩增进行快速微生物诊断

基本信息

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
项目类别：
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

项目摘要

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).

许多因素，尤其是抗病毒化疗的前景，已经需要快速，敏感，特定和实用的测定微生物诊断越来越明显。进度受到限制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通过聚合酶链反应为开发所需测定法。首席研究员及其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利用HIV-I作为原型剂。最近他们能够检测4个核苷酸突变的顺序外观逆转录酶基因，该基因是串行中AZT抗性的逆转录酶接受AZT治疗的患者的淋巴细胞样品。另一种已经开发了序列扩增的方法（37度C），单周期反应，可以在45中产生10（6）份分钟。还开发了基于珠子的三明治捕获测定法允许对该新产品的产品可重现检测和定量放大方案。该测定也适用于非分位检测。用于多种代理的同时测定的设备和方法也已经开发了单个标本。在此提案中，将优化方法，然后应用于四组目标生物：疱疹病毒，呼吸道病毒，生殖器病原体和眼科病原体。首席调查员有广泛的抗体，抗原和核酸的临床诊断经验酸。每个的大量临床标本的大量库存相关代理可以进行这些调查。一个将采用系统的，顺序的方法来实现以下内容目标： AIM 1：从目标生物中确定核酸序列提供生物体特异性，同时保持所有的反应性该生物的菌株。目标2：将特定的扩增和检测方法应用于临床已知包含目标剂（和控件）的材料以验证敏感性，特异性，再现性和可能的定量。 AIM 3：一旦针对目标集的单个代理进行测定已经开发了生物，应用塑料室或衍生物为临床综合征开发快速诊断方法的技术（呼吸道感染，生殖器病变，角膜结膜炎）。