A Kit for Rapid AST of Mycobacterium tuberculosis from Clinical Samples

描述（由申请人提供）：多药耐药（MDR-）和广泛耐药（XDR）结核病（TB）威胁着数十年的进步，使快速检测耐药性对结核病控制至关重要。近年来已经看到了直接从临床样品扩增和检测结核分枝杆菌（Mtb）核酸（NA）的核酸技术（NAT）的发展和部署。尽管NAT需要昂贵而复杂的仪器，但它的灵敏度与培养相当，并在数小时内而不是数天或数周内产生结果。然而，它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，多个单核苷酸多态性（SNP）必须同时扩增，检测和区分彼此，以鉴定耐药菌株。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，并被格式化为一个简单的执行试剂盒。SGM合成NA，即替代标记基因座（SML），作为抗生素对敏感生物体产生表型效应的替代标记。这使得NAT能够扩增和检测单个NA靶标，以确定Mtb对药物的敏感性，从而大大简化了基于NAT的耐药性检测。SGM通过重组分枝杆菌噬菌体传递给分枝杆菌，我们已经证明，重组分枝杆菌噬菌体可以快速报告培养的Mtb的抗生素敏感性谱。在本申请中，我们提出了三个目标，这将允许构建第二代SGM（2 <$SGM）报告噬菌体和能够检测每个样品</= 50 cfu的Mtb的测定。然后，我们将与分包商合作，创建一个研究试剂盒，以便于对数百个临床样本进行简化和可重复的检测，以确定检测试剂盒的检测时间以及其在确定 直接从新鲜临床分离株中获得完整的Mtb一线抗生素敏感性谱。构建2SGM报告噬菌体。Aim II.开发用于临床样本的标准化和可重复的一线抗生素敏感性测试（AST）的试剂盒。Aim III.直接从患者样本中检测TB AST的SGM-噬菌体试剂盒的临床评价。

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.