Twist-Sensor: Novel microarrays for multiplex detection of drug resistance

扭转传感器：用于多重检测耐药性的新型微阵列

• 批准号: 8648527
• 负责人: Alfredo Andres Celedon
• 金额: $ 29.99万
• 依托单位: SCANOGEN, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-15 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8648527
• 关键词: Acute; Bacteria; Bacterial Drug Resistance; Biological Assay; Blood; Caring; Cells; Cytolysis; DNA; DNA Microarray Chip; DNA amplification; Detection; Development; Devices; Diagnosis; Diagnostic; Diagnostic Procedure; Disease Outbreaks; Drug resistance; Electronics; Enterobacteriaceae; Equipment; Genes; Genetic Markers; Goals; Gram-Negative Bacteria; Hospitals; Hour; Human Resources; Image; Laboratories; Lactamase; Legal patent; Methods; Microbiology; Molecular Diagnosis; Noise; Nucleic Acids; Oligonucleotides; Organism; Pathogen detection; Patients; Phase; Preparation; Prevention; Procedures; Process; Resistance; Risk; Sampling; Sensitivity and Specificity; Stress; Techniques; Technology; Testing; Time; Training; base; carbapenem resistance; carbapenemase; improved; mortality; multiplex detection; new technology; novel; nucleic acid detection; public health relevance; rapid detection; sensor; single molecule; synthetic construct; tool

DESCRIPTION (provided by applicant): Drug-resistance is rapidly spreading among Gram-negative bacteria, seriously complicating the treatment of infected patients and increasing the risk of lethal outbreaks. In particular, carbapenem-resistant Enterobacteriaceae (CRE), organisms associated with high mortality rate (>40%), have become prevalent worldwide. During 2012, 18% of long-term acute care hospitals in the U.S had at least one case of CRE. Accurate and rapid identification of drug-resistance is fundamental for proper treatment and prevention of nosocomial spread. However, microbiology laboratories have deficient tools for resistance diagnosis. Phenotypic assays are the most common diagnostic methods, but they are slow (24 hours), frequently inconclusive and may fail to detect carbapenemases. Alternative methods based on nucleic acid detection exist, however, these methods are either time consuming and require trained personnel, or only cover a limited number of resistance determinants. Here, we will develop a novel class of microarrays based on Twist-Sensor technology for rapid, accurate and multiplex detection of drug resistance. Twist-Sensor is a novel technology for nucleic acid detection based on single molecule DNA hybridization and supercoiling. In this technique, the hybridized targets are subjected to disrupting torsional stres which allows for rapid detection of multiple target sequences with extremely low background noise using a simple device. We will demonstrate the capabilities of Twist-Sensor technology by developing a microarray able to detect multiple carbapenemase genes in a procedure with minimum hands- on time and with a total processing time of less than 2 hours. Aim 1 will demonstrate that Twist- Sensor technology is capable of detecting a resistance determinant in bacterial cultures without DNA amplification. Aim 2 will focus on developing a Twist-Sensor detection mechanism that is easy to automate, inexpensive and has high throughput. We will use this novel microarray to develop a multiplex assay for detection of the most prevalent carbapenemases (KPC, NDM, OXA-48, VIM and IMP). In order to optimize and characterize the functionality of the new microarray, we will analyze bacterial culture samples of resistant isolates.

描述(申请人提供)：抗药性正在革兰氏阴性细菌中迅速传播，使感染患者的治疗严重复杂化，并增加了致命疫情的风险。特别是，耐碳青霉烯类肠杆菌科(CRE)，与高死亡率(&gt；40%)相关的生物，已在世界范围内流行。2012年，美国18%的长期急性护理医院至少有一例CRE病例。准确、快速地识别耐药性是正确治疗和防止医院内传播的基础。然而，微生物学实验室缺乏耐药性诊断工具。表型分析是最常见的诊断方法，但它们速度慢(24小时)，经常不确定，而且可能无法检测出碳青霉烯酶。基于核酸检测的替代方法已经存在，然而，这些方法要么耗时且需要训练有素的人员，要么仅涵盖有限数量的抗性决定因素。在这里，我们将开发一种基于扭转传感器技术的新型微阵列，用于快速、准确和多重检测耐药。扭转传感器是一种基于单分子DNA杂交和超螺旋技术的新型核酸检测技术。在这种技术中，杂交目标受到破坏性的扭转应力，这允许使用简单的设备快速检测具有极低背景噪声的多个目标序列。我们将通过开发一种微阵列来展示扭转传感器技术的能力，该微阵列能够在最短的动手时间和总处理时间不到2小时的过程中检测多个碳青霉烯酶基因。目的1将证明扭转传感器技术能够在不进行DNA扩增的情况下检测细菌培养中的耐药性决定簇。AIM 2将专注于开发一种易于自动化、成本低廉、产量高的扭转传感器检测机制。我们将利用这种新型的微阵列来建立一种检测最流行的碳青霉烯酶(KPC、NDM、OXA-48、VIM和IMP)的多重方法。为了优化和表征新的微阵列的功能，我们将分析耐药菌株的细菌培养样本。