Bacillus anthracis Detection with RNA Microchip

描述(由申请人提供)： 三年前，炭疽芽孢杆菌被生物恐怖分子用来攻击公民和政府机构。炭疽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，而且它的孢子形态对灭活具有高度的抵抗力。如果不及时治疗，炭疽吸入和胃肠道感染最有可能导致死亡。为确保立即就医，在现场和临床环境中快速准确地检测炭疽杆菌是至关重要的。为了满足这一需求，我们通过RNA-DNA杂交模板结合、RNaseH消化和Klenow 3‘-延伸/标记，开发了一种直接检测总RNA样本中特定RNA的系统。检测灵敏度可达全摩尔(10[-18]摩尔)水平。这种以微板形式检测RNA的新方法不需要反转录、聚合酶链式反应、激光激发、荧光检测、转录和/或凝胶电泳。为了建立一种灵敏和特异的病原体检测策略，我建议在这个RNA 3‘标记/检测系统的基础上，通过将识别炭疽杆菌特征RNA的RNA-DNA杂交模板固定在一个微芯片上来开发一种RNA微芯片方法和技术。简而言之，标志性的RNA被杂交到这个微芯片上，而非特定的RNA被冲走。结合的RNA通过RNaseH酶切和Klenow标记，在芯片上用生物素标记。然后用抗生素抗体-碱性磷酸酶(AP)结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，通过在该RNA芯片上进行签名RNA检测，炭疽芽孢杆菌可以得到阳性鉴定。对检测系统进行优化后，可在20分钟或更短的时间内完成病原菌的检测。这种简单快速的RNA微芯片技术还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、医疗保健诊断、SNP快速分析和个性化用药、癌症检测和亚型分类以及食品、空气和水的安全监测。这些长期目标超出了这项提议的范围。

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.