A Kit for Rapid AST of Mycobacterium tuberculosis from Clinical Samples

描述(申请人提供)：多药耐药(MDR-)和广泛耐药(XDR)结核病(TB)有可能破坏数十年的进展，使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)的发展和部署见证了直接从临床样本中扩增和检测结核分枝杆菌(MTB)核酸(NA)的技术。尽管NAT需要昂贵和复杂的仪器，但它们表现出与培养一样的敏感性，并且在几小时内就能产生结果，而不是几天或几周。然而，它们的成本使其无法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(SNP)。为了克服这些限制，我们设计了一种新型的分子报告系统，SML生成模块(SGM)，它可以确定对任何药物的耐药性，并被格式化为一个易于执行的试剂盒。SGM合成了一种NA，即代理标记基因座(SML)，作为抗菌剂对敏感生物施加的表型效应的代理标记。这使得NAT可以放大和检测单个NA靶标，以确定结核分枝杆菌对药物的敏感性，从而极大地简化了基于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，这种噬菌体可以快速报告培养的结核分枝杆菌的抗生素敏感性图谱。在本申请中，我们提出了三个目标，使S能够构建第二代SGM(2？SGM)报告噬菌体和能够检测每个样本50cfu的Mtb的方法。然后，我们将与分包商合作创建一套研究试剂盒，促进对数百个临床样本进行简化和可重复的测试，以确定检测化验的时间以及其在确定 直接从新鲜的临床分离株中直接完成结核分枝杆菌的一线药敏图谱。目的I.构建2？SGM报告噬菌体。目的II.临床标本一线抗生素药敏试验试剂盒的研制。目的III.直接从患者标本中提取结核分枝杆菌门冬氨酸转氨酶的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.