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

描述（由申请人提供）：耐药性在革兰氏阴性菌中迅速蔓延，严重复杂化了受感染患者的治疗，并增加了致命爆发的风险。特别是碳青霉烯耐药肠杆菌科（CRE），这种与高死亡率相关的生物（bbb40 %）已在世界范围内普遍存在。2012年，美国18%的长期急症护理医院至少有一例CRE。准确和快速地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），往往不确定，可能无法检测到碳青霉烯酶。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训练有素的人员，要么只能覆盖有限数量的耐药决定因素。在这里，我们将开发一种基于扭曲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杂交和超卷曲技术的新型核酸检测技术。在该技术中，杂交目标受到破坏扭转应力，从而允许使用一个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Twist-Sensor技术的能力，该微阵列能够在最少的手工时间和少于2小时的总处理时间内检测多个碳青霉烯酶基因。目的1将证明扭曲传感器技术能够在没有DNA扩增的情况下检测细菌培养中的抗性决定因素。目标2将侧重于开发一种易于自动化，廉价且具有高吞吐量的扭曲传感器检测机制。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）。为了优化和表征新的微阵列的功能，我们将分析耐药菌株的细菌培养样本。

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.