Bacillus anthracis Detection with RNA Microchip

描述（由申请人提供）： 炭疽杆菌三年前被生物恐怖分子用来攻击民间和政府机构。B。炭疽确实是一种潜在的生物战剂，因为它具有高度致病性，易于培养和生产，而且其孢子形式对灭活具有高度抗性。B。如果感染者得不到及时治疗，吸入炭疽病和胃肠道感染极有可能导致死亡。确保及时就医，快速准确检测B。炭疽病在现场和临床环境中是必不可少的。为了满足这一需求，我们开发了一种通过RNA-DNA杂交模板结合、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。其检测灵敏度可达阿摩尔（10 ~（-18）mole）级。这种以微孔板形式进行RNA检测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。为了开发一种灵敏和特异的病原体检测策略，我在这里建议开发一种基于这种RNA 3 '-标记/检测系统的RNA微芯片方法和技术，通过固定识别B的RNA-DNA杂交寺庙。微芯片上的炭疽菌标志RNA。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，在该芯片上用生物素标记结合的RNA。然后，生物素标记物与抗生物素蛋白抗体-碱性磷酸酶（AP）结合物一起转化为酶标记物。最后，通过AP催化的荧光反应和化学发光检测来检测标记的RNA。因此，炭疽芽孢杆菌可以通过该RNA微芯片上的特征RNA检测来阳性鉴定。通过对检测系统的优化，可以在20分钟或更短的时间内实现病原体的检测。这种简单快速的RNA微芯片技术也将在未来用于生物防御和临床环境中的多种病原体和疾病检测，用于即时诊断，用于快速SNP分析和个性化药物管理，用于癌症检测和亚型分类，以及用于食品，空气和水安全监测。这些长期目标超出了本提案的范围。

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.