MOLECULAR PROBES FOR DIAGNOSIS OF COCCIDIOIDOMYCOSIS

球孢子菌病病例数的大幅增加（谷 发烧），一种人类的真菌呼吸道疾病， 美国西南部近五年来我们的目标是 开发快速和相对简单的方法来诊断这种疾病 疾病目前的方法是不够的，因为它们主要是 取决于患者抗体检测（抗体产生可能 在免疫功能低下的患者中延迟或减弱），并且因为 目前用于抗体检测的多组分抗原 批次之间存在差异，并显示出一些交叉反应性 异源血清（假阳性反应）。政府不得不建议 球孢子菌病三种诊断方法的发展， 设计了实验来测试特异性和敏感性， 分子探测器。我们将使用一种重组补体 特异性抗体标准免疫测定中的固定抗原（CF-Ag） 检测，CF-Ag和球孢子菌特异性单克隆抗体 抗原（CS-Ag）检测抗原血症，并建立了PCR方法 在PI。的实验室对C.患者体液中的免疫DNA检测。 多种对照和阳性患者液体（> 400份血清、CSF、尿液， BALs和痰液）将用于 开放和盲法研究，以测试我们的诊断 方法. 建议的商业应用：该建议概述了以下方法： 检测（a）病原体特异性抗体，（B）抗原血症和（c）DNA 在患者血清和其它体液中使用（1）细菌产生的 重组抗原，（2）单克隆抗体和（3）聚合酶链 来自病原体特异性基因的PCR反应引物对， 分别 每种试剂（分子探针）都可以在 无限数量，可以存储无损失的活动无限 时间，可以标准化（质量控制），可以合并 用于临床应用的单个检测试剂盒， 临床实验室的检测方法。

A major increase in the number of cases of coccidioidomycosis (Valley fever), a fungal respiratory disease of humans, has occurred in southwestern United States during the last five years. Our goals are to develop rapid and relatively simple methods for diagnosis of this disease. Current methods are inadequate because they are primarily dependent on patient antibody detection (antibody production may be delayed or attenuated in immunocompromised patients), and because multicomponent antigens currently used for antibody detection are variable between batches and display some cross-reactivity with heterologous sera (false positive reactions). We have proposed the development of three approaches to diagnosis of coccidioidomycosis and have designed experiments to test specificity and sensitivity of the molecular probes to be used. We will employ a recombinant, complement fixation antigen (CF-Ag) in standard immunoassays for specific antibody detection, monoclonal antibody to the CF-Ag and Coccidioides-specific antigen (CS-Ag) for detection of antigenemia, and a PCR method developed in the P.I.'s laboratory for C. immitis DNA detection in patient fluids. Multiple control and positive patient fluids (>400 sera, CSF, urine, BALs, and sputum) submitted by 8 physician-consultants will be used in open and blind studies for testing the efficacy of our diagnostic methods. PROPOSED COMMERCIAL APPLICATION: This proposal outlines methods for detection of (a) pathogen-specific antibody, (b) antigenemia and (c) DNA in patient sera and other body fluid using (1) bacterial-produced recombinant antigen, (2) monoclonal antibodies and (3) a polymerase chain reaction (PCR) primer-pair derived from a pathogen-specific gene, respectively. Each reagent (molecular probe) can be produced in unlimited amounts, can be stored without loss of activity for unlimited time, can be standardized (quality-controlled), and can be incorporated into individual test kits for clinical application using established testing methods in clinical laboratories.