Integrated Immunoassay/PCR Test for Bioterrorism Agents

描述（由研究者提供）：这项小型企业创新研究建议将三种不同技术的最佳特征：免疫测定，细胞培养和实时聚合酶链反应（PCR）结合到一个用于CDC A类试剂环境检测的综合测试中。第一阶段将包括生物传感器仪器、测试盒、试剂和测定的开发。第一阶段将使用一种生物恐怖剂-炭疽孢子。申报的三步生物传感器将具有以下特性：在20分钟内检测病原体的快速、灵敏的免疫测定，验证病原体活力的细胞培养步骤，确认阳性免疫测定结果的实时PCR步骤，定量样品中生物体或毒素分子的数量，一个紧凑的和独立的一次性测试盒，安全地限制环境样品和所有测试副产品，自动化样品制备和数据采集，综合测试将同时比目前的方法更敏感和更具体，无论是现场快速测试还是实验室应用。通过从环境样品中选择性地捕获致病性炭疽孢子并将其从样品中的所有其他生物体中纯化，生物传感器可以为随后的PCR反应提供不含抑制剂的孢子衍生的营养细胞裂解物。该测试解决了影响其他方法的假阴性（灵敏度，活力，抑制剂）和假阳性（交叉反应，污染）的根本原因。因为它证实了生物的生存能力，该测试将使真实的时间监测去污工作，如最近在华盛顿邮政设施和政府办公室进行。

DESCRIPTION (provided by investigator): This Small Business Innovation Research proposes to combine the best features of three different technologies: immunoassay, cell culture and real-time polymerase chain reaction (PCR), into one integrated test for environmental detection of the CDC Category A agents. Phase I will comprise development of the biosensor instrument, the test cartridge, reagents, and the assays. One bioterrorism agent, anthrax spores, will be used in Phase I. The proposed Three-Step Biosensor will have the following properties: a rapid, sensitive immunoassay that detects pathogens within 20 minutes, a cell culture step that verifies the viability of the pathogens, a real-time PCR step that confirms positive immunoassay results, quantitation of the number of organisms or toxin molecules in the sample, a compact and self-contained disposable test cartridge that safely confines the environmental sample and all test by-products, automated sample preparation and data acquisition, and a portable instrument format for use in the field by first responders The integrated test will be simultaneously more sensitive and more specific than current methods, both for on-site rapid tests and laboratory applications. By selectively capturing pathogenic anthrax spores from environmental samples and purifying them from all other organisms in the sample, the biosensor can provide a spore-derived vegetative cell lysate free of inhibitors for subsequent PCR reactions. The test addresses the root causes of both false negatives (sensitivity, viability, inhibitors) and false positives (cross-reaction, contamination) that affect other methods. Because it confirms organism viability, the test will enable real time monitoring of decontamination efforts, such as that recently performed at Washington DC postal facilities and government offices.