A Kit for Rapid AST of Mycobacterium tuberculosis from Clinical Samples

从临床样本中快速检测结核分枝杆菌 AST 的试剂盒

• 批准号: 8436284
• 负责人: Matthew Charles Mulvey
• 金额: $ 23.1万
• 依托单位: SEQUELLA, INC.
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-01 至 2014-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8436284
• 关键词: Antibiotic susceptibility; Antibiotics; Antimicrobial Effect; Antitubercular Agents; Antitubercular Antibiotics; Area; Bacteria; Bacteriophages; Biological Assay; Biological Markers; Clinical; Clinical Research; Colony-forming units; Communicable Diseases; Complex; Detection; Development; Diagnosis; Disease Resistance; Drug resistance; Drug resistance in tuberculosis; Drug usage; Ensure; Equilibrium; Evaluation; Extreme drug resistant tuberculosis; Future; Gene Mutation; Generations; Genetic; Genus Mycobacterium; Goals; Hour; Infection; Infectious Diseases Research; International; Knowledge; Marketing; Medical; Methods; Microscopy; Molecular; Multi-Drug Resistance; Mycobacteriophages; Mycobacterium tuberculosis; Nucleic Acids; Organism; Patients; Peripheral; Peru; Pharmaceutical Preparations; Physicians; Predisposition; Procedures; Reaction; Recombinants; Regimen; Reporter; Reporting; Reproducibility; Research; Research Design; Resistance; Rifampin; Sampling; Sensitivity and Specificity; Single Nucleotide Polymorphism; Site; South Korea; Specimen; Sputum; Surrogate Markers; System; Technology; Testing; Time; Tuberculosis; Work; base; cost; design; effective therapy; instrumentation; isoniazid; killings; new technology; novel; prevent; public health relevance; rapid detection; research clinical testing; research study; resistance mutation; resistant strain; tool; tuberculosis drugs; tuberculosis treatment

DESCRIPTION (provided by applicant): Multi-drug resistant (MDR-) and extensively-drug resistant (XDR) Tuberculosis (TB) threaten to undo decades of progress, making the rapid detection of drug resistance crucial to TB control. Recent years have seen the development and deployment of nucleic acid technologies (NAT) that amplify and detect Mycobacterium tuberculosis (Mtb) nucleic acid (NA) directly from clinical samples. Although they require expensive and sophisticated instrumentation, NATs demonstrate sensitivities on par with culture, and yield results within hours rather than days or weeks. However, their cost prevents deployment to the peripheral labs where most patients seek diagnosis and treatment. In addition, multiple single nucleotide polymorphisms (SNP) must be simultaneously amplified, detected and discriminated from each other in order to identify resistant strains. To overcome these limitations, we designed a novel molecular reporter system, the SML-Generation Module (SGM), which can determine resistance to any drug and be formatted as a simple-to-execute kit. The SGM synthesizes a NA, the Surrogate Marker Locus (SML), as a surrogate marker for the phenotypic effects antimicrobials exert on susceptible organisms. This allows NATs to amplify and detect a single NA target to determine susceptibility of Mtb to a drug, dramatically simplifying NAT-based detection of drug resistance. The SGM is delivered to Mycobacteria by a recombinant mycobacteriophage, which we have shown can rapidly report the antibiotic susceptibility profile of cultured Mtb. In this application, we propose three Aims that will allow s to construct a second generation SGM (2¿SGM) reporter phage and assay capable of detecting </= 50cfu of Mtb per sample. We will then work with a subcontractor to create a research kit facilitating streamlined and reproducible testing of hundreds of clinical samples to establish the time to detection of the assay as well as its sensitivity and specificity in the determination of a complete front-line antibiotic susceptibility profile of Mtb directly from fresh clinical isolates.Aim I. Construction of a 2¿SGM reporter phage. Aim II. Development of a kit for standardized and reproducible front-line antibiotic susceptibility testing (AST) of clinical samples. Aim III. Clinial Evaluation of the SGM-phage kit for TB AST directly from patient samples.

描述（由申请人提供）：耐多药（MDR-）和广泛耐药（XDR）结核病（TB）有可能使数十年的进展付之一篑，这使得快速检测耐药性对结核病控制至关重要。近年来，核酸技术（NAT）的发展和部署，可以直接从临床样本中扩增和检测结核分枝杆菌（Mtb）核酸（NA）。虽然它们需要昂贵和复杂的仪器，但NATs显示出与培养相当的灵敏度，并且在几小时内产生结果，而不是几天或几周。然而，他们的成本阻碍了部署到大多数患者寻求诊断和治疗的外围实验室。此外，为了鉴定耐药菌株，必须同时扩增、检测和区分多个单核苷酸多态性（SNP）。为了克服这些限制，我们设计了一种新的分子报告系统，sml生成模块（SGM），它可以确定对任何药物的耐药性，并被格式化为一种简单易用的试剂盒。SGM合成了一个NA，即替代标记位点（SML），作为抗菌剂对敏感生物的表型效应的替代标记。这使得NATs能够扩增和检测单个NA靶点以确定结核分枝杆菌对药物的敏感性，从而大大简化了基于NA的耐药性检测。SGM通过重组的分枝杆菌噬菌体传递给分枝杆菌，我们已经证明它可以快速报告培养的结核分枝杆菌的抗生素敏感性。在本应用中，我们提出了三个目标，这将允许我们构建第二代SGM（2¿SGM）报告噬菌体和能够检测每个样品</= 50cfu Mtb的检测方法。然后，我们将与分包商合作创建一个研究工具包，方便对数百个临床样本进行简化和可重复的测试，以确定检测检测的时间以及测定a的敏感性和特异性