Bioagent Characterization by Iterative Extension

描述(申请人提供)：项目摘要-提出了一种新的基于症状的诊断系统，用于快速检测和鉴定B类和C类优先病原体。第一阶段将专注于霍乱弧菌基因和来自类似病原体的基因。该技术依赖于解旋酶驱动的迭代引物延伸反应(HDIE)，该反应使用等温、多路目标扩增来产生带有代表样本DNA的序列的短的、标记的单链DNA产物。这些反应产物非常适合与固定在微阵列上的短探针杂交，从而提供样本中任何病原体的基因鉴定。第二阶段将把这项技术扩展到各种B类和C类优先病原体。这项研究的成功完成将产生一个基于症状的诊断系统，该系统可以在大量候选样本中识别出一种或多种病原体。这项技术将是等温的，需要最少的初始反应设备，使其在生物武器攻击或疫情爆发时对第一反应人员有用。 HDIE依赖于这样一个事实，即在12到40个碱基的跨度内，任何基因中都有大量区域缺少四个DNA碱基中的一个。HDIE引物被设计成与紧邻这些空白区之一的3‘端的一段靶DNA互补。一旦该引物与目标DNA相结合，延伸反应就从该引物的3‘端开始，使用缺少A、T、G或C的区域作为模板。当第一次遇到缺失碱基时，通过掺入互补的双脱氧核苷酸终止延伸反应。DNA解旋酶将延伸反应产物从模板分子中分离出来，允许迭代的非指数靶扩增。HDIE产物可以与微阵列探针杂交，并使用传统的荧光方法进行检测。反应在很短的时间内终止，加上扩增是线性的而不是指数的，这一事实允许在一个反应管中存在数百个引物。 尽管HDIE方法旨在克服并行病原体诊断所特有的困难，但它可以扩展到许多进入利润丰厚的分子诊断市场的应用，包括食源性病原体检测、农业和兽医测试、水测试和临床测试。2005年，全球分子诊断市场预计将达到65亿美元，约占整个诊断市场的3.3%，约占体外诊断市场的14%。分子诊断市场预计到2010年将增加到120亿美元，到2015年将增加到350亿美元。 相关性--拟议的研究直接解决了对基于症状的分析的需求，该分析可以在生物武器攻击或重大疫情爆发的情况下快速识别导致疾病的一个或多个病原体。

DESCRIPTION (provided by applicant): Project Summary - A new syndrome-based diagnostic system for rapid detection and identification of category B and C priority pathogens is proposed. Phase 1 will focus on V. cholerae genes and genes from similar pathogens. The technique relies upon a helicase-driven iterative primer extension reaction (HDIE) that uses isothermal, multiplexed target amplification to produce short, labeled, single-stranded DNA products with sequences representative of the sample DNA. These reaction products are ideal for hybridization to short probes immobilized on microarrays, thus providing gene identification of any pathogen in the sample. Phase II will expand the technique to a wide variety of category B and C priority pathogens. Successful completion of the research will result in a syndrome-based diagnostic system that can identify one or more pathogens in a sample among a large number of candidates. The technique will be isothermal and require minimum equipment for the initial reactions, making it useful to first responders in a biowarfare attack or an outbreak. HDIE relies on the fact that there are a large number of regions in any gene that are missing one of the four DNA bases over a span of 12 to 40 bases. HDIE primers are designed to be complementary to a section of the target DNA immediately adjacent to the 3' end of one of these void regions. Once the primer is annealed to the target DNA, the extension reaction proceeds from the 3' end of the primer using the region lacking either A, T, G, or C as the template. When the first occurrence of the missing base is encountered the extension reaction is terminated by incorporation of a complementing dideoxynucleotide. DNA helicases separate the extension reaction products from the template molecules, allowing iterative non-exponential target amplification. The HDIE products can be hybridized to microarray probes and detected using conventional fluorescence methods. The fact that the reaction is terminated after a very short extension, along with fact that amplification is linear rather than exponential, allows hundreds of primers to be present in a single reaction tube. Although the HDIE approach is designed to overcome difficulties specific to parallel pathogen diagnosis, it can be extended to many applications that tap into the lucrative molecular diagnostics market, including foodborne pathogen detection, agricultural and veterinary testing, water testing, and clinical testing. In 2005, the global market for molecular diagnostics is expected to be $6.5 billion, or about 3.3% of the total diagnostics market and approximately 14% of the in vitro diagnostic market. The molecular diagnostics market is forecast to increase to $12 billion by 2010 and $35 billion by 2015. Relevance - The proposed research directly addresses the need for a syndrome-based assay that can rapidly identify' the pathogen or pathogens responsible for disease in case of a biowarfare attack or a major outbreak.