Rapid ID and AST Directly from Whole Blood Using Single Molecule Detection

项目摘要 血流病原体的标准鉴定（ID）和抗菌药物敏感性试验（AST）是 使用需要血液培养的缓慢方法进行。诊断结果可在三天或更长时间内获得 在诊断过程开始之后。快速诊断结果的缺乏迫使医生治疗患者， 广谱抗生素对越来越多的耐药微生物无效， 导致更多的抗菌素耐药性的产生。在这里，我们建议快速发展 诊断平台，如果成功，将能够有效和有针对性地治疗血流感染患者 几小时内 新平台将使用简单而强大的单分子检测直接分析全血样本 这种方法被称为单分子拴系或SMOLT。该平台将是快速的（ID在1小时内，AST在4- 6小时），易于使用，具有成本效益。我们的初步结果表明，SMOLT多重检测可以检测 血液中的细菌，检测限（LOD）在1-10 CFU/mL之间，周转时间约为1小时。 同样的技术也可以用于AST。初步结果显示，用抗生素短暂孵育 随后进行SMOLT检测可用于确定菌株在高浓度下是敏感的、中间的还是抗性的。 与指定的类别一致，可作为标准参考方法。在目标1中，我们提出发展 能够检测和鉴定6种高度相关病原体的rRNA的SMOLT多重ID检测试剂盒 直接在全血中。目的2的目标是开发用于本发明的组合的SMOLT表型AST测定。 6种鉴别种和14种相关药物。我们的第三个目标是为SMOLT ID开发一个原型仪器， AST检测试剂盒易于使用，操作时间最短。最后一个目的是评价ID和AST测定 使用接种有新鲜和储备临床分离物以及模式菌株的组合的血液样品。

Project Summary Standard identification (ID) and antimicrobial susceptibility testing (AST) of bloodstream pathogens are conducted using slow methods that require blood culture. Diagnostic results are available three or more days after the diagnostic process is started. The lack of rapid diagnostic results forces physicians to treat patients with broad-spectrum antibiotics which are not effective against a growing number of resistant organisms and can induce the development of more antimicrobial resistance. Here, we propose the development of a rapid diagnostic platform that, if successful, will enable effective and targeted therapy of bloodstream infection patients within hours. The new platform will directly analyze whole blood samples using a simple and robust single molecule detection approach called Single MOlecule Tethering or SMOLT. The platform will be rapid (ID in one hour and AST in 4- 6 hours), easy-to-use, and cost-effective. Our preliminary results show that a SMOLT multiplex assay can detect bacteria in blood with limits of detection (LOD) between 1-10 CFU/mL with a turn-around-time of about an hour. The same technique can be used for AST. Preliminary results show that a brief incubation with an antibiotic follow by SMOLT detection can be used to determine if a strain is susceptible, intermediate or resistance in high agreement with the category assigned be a standard reference method. In aim 1, we propose the development of a SMOLT multiplex ID assay capable of detecting and identifying the rRNA of six highly relevant pathogens directly in whole blood. The goal of aim 2 is to develop a SMOLT phenotypic AST assay for combinations of the six ID species and 14 relevant drugs. Our third aim is to develop a prototype instrument for the SMOLT ID and AST assays that is easy-to-use with minimum hands-on-time. The last aim is to evaluate the ID and AST assays using blood samples seeded with a combination of fresh and stock clinical isolates as well as type strains.