Bacillus anthracis Detection with RNA Microchip

RNA 微芯片检测炭疽杆菌

• 批准号: 7140518
• 负责人: ZHEN HUANG
• 金额: $ 14.11万
• 依托单位: GEORGIA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7140518
• 关键词: Bacillus anthracis; bacterial RNA; biohazard detection; bioterrorism /chemical warfare; immobilized enzymes; microarray technology; nucleic acid hybridization; ribonuclease H; technology /technique development

DESCRIPTION (provided by applicant): Bacillus anthracis was used by bio-terrorists to attack the civil and governmental institutions three years ago. B. anthracis is indeed a potential biological warfare agent because it is highly pathogenic and easy to cultivate and produce, and because its spore form is highly resistant to inactivation. B. anthracis inhalation and gastrointestinal infection most likely lead to death if the infected patients are not treated promptly. To ensure immediate medical treatment, rapid and accurate detection of B. anthracis in field and clinical settings is essential. To meet the need, we have developed a direct detection system for specific RNAs in a total RNA sample via RNA-DNA hybrid template binding, RNase H digestion, and Klenow 3'- extension/labeling. The detection sensitivity can reach up to attomole (10[-18 ]mole) level. This novel approach of RNA detection in microplate format doesn't require reverse transcription, polymerase chain reaction, laser excitation, fluorescence detection, transcription, and/or gel electrophoresis. To develop a sensitive and specific pathogen detection strategy, I propose here to develop an RNA microchip methodology and technology on the base of this RNA 3'-labeling/detection system, by immobilizing the RNA-DNA hybrid temples that recognize B. anthracis signature RNAs on a microchip. In brief, the signature RNAs are hybridized to this microchip while non-specific RNAs are washed away. The bound RNAs are labeled with biotin on this chip via RNase H digestion and Klenow labeling. The biotin labels are then converted to the enzyme labels with the antibiotin antibody-alkaline phosphatase (AP) conjugate. Finally, the labeled RNAs are detected by the AP-catalyzed chemiluminescent reaction and chemiluminescence detection. Bacillus anthracis can thus be positively identified via signature RNA detection on this RNA microchip. After optimization of the detection system, the pathogen detection can be achieved in twenty minutes or shorter time. This simple and rapid RNA microchip technology will also be used in the future for multiple pathogen and disease detection in biodefense and in clinical settings, for point-of-care diagnosis, for rapid SNP analysis and individualized medicine administration, for cancer detection and subtype classification, and for food, air and water safety monitoring. These long-term goals are beyond the scope of this proposal.

描述（由申请人提供）： 三年前，生物恐怖分子利用炭疽杆菌攻击民间和政府机构。炭疽芽孢杆菌确实是一种潜在的生物战剂，因为它具有高致病性，易于培养和生产，而且其孢子形式对灭活具有很强的抵抗力。如果感染者不及时治疗，炭疽杆菌吸入和胃肠道感染很可能导致死亡。为了确保立即就医，在现场和临床环境中快速、准确地检测炭疽杆菌至关重要。为了满足这一需求，我们开发了一种通过 RNA-DNA 混合模板结合、RNase H 消化和 Klenow 3'- 延伸/标记来直接检测总 RNA 样品中特定 RNA 的系统。检测灵敏度可达阿托摩尔(10[-18]摩尔)水平。这种以微孔板形式检测 RNA 的新方法不需要逆转录、聚合酶链式反应、激光激发、荧光检测、转录和/或凝胶电泳。为了开发灵敏且特异的病原体检测策略，我在此建议通过固定识别炭疽芽孢杆菌特征 RNA 的 RNA-DNA 混合模板，开发一种基于 RNA 3' 标记/检测系统的 RNA 微芯片方法和技术在微芯片上。简而言之，特征 RNA 与该微芯片杂交，而非特异性 RNA 被洗掉。通过 RNase H 消化和 Klenow 标记，结合的 RNA 在该芯片上被生物素标记。然后使用抗生物素抗体-碱性磷酸酶 (AP) 缀合物将生物素标记转化为酶标记。最后，通过AP催化的化学发光反应和化学发光检测来检测标记的RNA。因此，可以通过该 RNA 微芯片上的特征 RNA 检测来明确识别炭疽杆菌。检测系统优化后，可以在二十分钟或更短的时间内实现病原体检测。这种简单快速的RNA微芯片技术未来还将用于生物防御和临床环境中的多种病原体和疾病检测、即时诊断、快速SNP分析和个体化用药、癌症检测和亚型分类，以及食品、空气和水安全监测。这些长期目标超出了本提案的范围。

项目成果

Direct and rapid detection of RNAs on a novel RNA microchip.

• DOI: 10.1002/cbic.201000170
• 发表时间: 2010-07-05
• 期刊: CHEMBIOCHEM
• 影响因子: 3.2
• 作者: Spencer, Sarah M.;Lin, Lina;Chiang, Cheng-Feng;Peng, Zhengchun;Hesketh, Peter;Salon, Jozef;Huang, Zhen
• 通讯作者: Huang, Zhen