AMPLIFIED NUCLEIC ACID-EIA FOR MICROBIAL DIAGNOSIS

用于微生物诊断的扩增核酸-EIA

基本信息

批准号：
2065609
负责人：
ROBERT H YOLKEN
金额：
$ 21.9万
依托单位：
JOHNS HOPKINS UNIVERSITY
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

来源：
https://reporter.nih.gov/project-details/2065609
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

项目摘要

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很重要病患者和疾病暴发的遏制。最多以前开发的测定系统使用了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可用于使用微生物诊断；但是，没有一种方法证明没有一种方法是理想的选择检测所有微生物剂。我们建议将固相结合捕获，核酸扩增和免疫测定方法开发单个测定系统以检测微生物病原体。靶核酸将通过与RNA探针的杂交。去除污染核酸和其他材料通过洗涤，结合的核酸将通过周期性酶促反应洗脱并放大。这然后将通过固相定量扩增的核酸酶免疫测定程序。这种测定格式将允许特异性微生物基因组的敏感扩增和检测人体液体中的序列，而无需干扰材料。此外，利用熟悉的酶免疫测定反应定量程序将允许利用在广泛的临床和研究实验室中的测定系统环境。将开发捕获放大测定法以检测肠道，呼吸和性传播的致病剂疾病。最初的靶向生物将包括轮状病毒，流感病毒和沙眼衣原体。灵敏度和特异性测定将使用纯化的核酸和A以逐步的方式进行评估同源和异源微生物的范围。临床然后将评估测定系统的效率和特异性利用从定义明确的研究人群获得的连续样品。这这些测定的成功开发将随后开发其他微生物剂的类似测定法。生产力这些测定法可能会改善患者的医疗服务可疑感染并最大程度地减少医院内疾病的传播环境。