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（由申请人提供）：耐药性正在革兰氏阴性细菌中迅速蔓延，使感染患者的治疗严重复杂化，并增加了致命爆发的风险。特别是具有高死亡率（>40%）的耐碳青霉烯类肠杆菌科细菌（CRE）已在全球范围内流行。 2012 年，美国 18% 的长期急症护理医院至少发生过 1 例 CRE 病例。准确、快速识别耐药性是正确治疗和预防院内传播的基础。然而，微生物实验室缺乏耐药性诊断工具。表型测定是最常见的诊断方法，但速度较慢（24 小时），常常无法得出结论，并且可能无法检测碳青霉烯酶。存在基于核酸检测的替代方法，然而，这些方法要么耗时且需要经过培训的人员，要么仅涵盖有限数量的耐药决定因素。在这里，我们将开发一类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性。 Twist-Sensor是一种基于单分子DNA杂交和超螺旋的核酸检测新技术。在这项技术中，杂交的目标受到破坏性扭转应力的作用，从而可以使用简单的设备以极低的背景噪声快速检测多个目标序列。我们将通过开发一种能够检测多种碳青霉烯酶基因的微阵列来展示扭转传感器技术的功能，该微阵列的操作时间最少，总处理时间不到 2 小时。目标 1 将证明 Twist-Sensor 技术能够在不扩增 DNA 的情况下检测细菌培养物中的耐药决定簇。目标 2 将重点开发一种易于自动化、廉价且具有高吞吐量的扭转传感器检测机制。我们将使用这种新型微阵列开发一种多重检测方法，用于检测最常见的碳青霉烯酶（KPC、NDM、OXA-48、VIM 和 IMP）。为了优化和表征新微阵列的功能，我们将分析耐药菌株的细菌培养样本。