Bacillus anthracis Detection with RNA Microchip

RNA 微芯片检测炭疽杆菌

• 批准号: 6983584
• 负责人: ZHEN HUANG
• 金额: $ 25.46万
• 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 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6983584
• 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，生物恐怖主义者使用了炭疽芽孢杆菌，以攻击民事和政府机构。 B.炭疽病确实是一种潜在的生物战剂，因为它具有高度致病性且易于培养和产生，并且由于其孢子形式对失活具有高度抗性。 B.炭疽病吸入和胃肠道感染，如果未迅速治疗感染患者，很可能会导致死亡。为了确保立即治疗，至关重要的是，在野外和临床环境中快速准确检测炭疽病。为了满足需求，我们通过RNA-DNA杂交模板结合，RNase H Digestion和Klenow 3'-扩展/标记开发了总RNA样品中特定RNA的直接检测系统。检测灵敏度可以达到attomole（10 [-18] mole）水平。这种新颖的RNA检测方法以微板格式检测不需要逆转录，聚合酶链反应，激光激发，荧光检测，转录和/或凝胶电泳。为了制定一种敏感而特定的病原体检测策略，我在这里建议通过固定识别植物芽孢杆菌的RNA-DNA混合神庙在此RNA 3'Labeling/检测系统的基础上开发RNA微芯片方法论和技术，该rna-dna杂交庙可以在微芯片上识别B. anthracis Signature RNA。简而言之，将签名RNA与该微芯片杂交，而非特异性RNA被冲走。通过RNase H Digestion和Klenow标记，将结合的RNA用生物素标记为生物素。然后将生物素标记转换为抗蛋白抗体 - 碱性磷酸酶（AP）结合物转换为酶标记。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检测来正面鉴定炭疽芽孢杆菌。优化检测系统后，可以在二十分钟或更短的时间内实现病原体检测。将来，这种简单而快速的RNA微芯片技术也将用于生物幻影和临床环境中的多种病原体和疾病检测，用于保健点诊断，快速SNP分析和个性化医学给药，用于癌症检测和亚型分类，以及食品，空气，空气和水安全监测。这些长期目标超出了该提议的范围。