Inexpensive multiplexed ligation probe assay to genotype drug resistant M.tubberc

廉价的多重连接探针测定耐药结核分枝杆菌基因型

• 批准号: 8315433
• 负责人: Heather Koshinsky
• 金额: $ 24.8万
• 依托单位: INVESTIGEN, 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 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8315433
• 关键词: Automation; Biological Assay; Characteristics; Chemistry; Clinical; Collection; Communities; DNA; Data; Detection; Development; Diagnosis; Diagnostic; Diagnostic tests; Disease; Drug Resistant Tuberculosis; Drug resistance; Drug-sensitive; Early Diagnosis; Equipment; Extreme drug resistant tuberculosis; FDA approved; Gene Mutation; Genes; Genotype; Goals; Hand; Hour; Image; Knowledge; Left; Ligase; Ligation; Link; Location; Methods; Microscopy; Mission; Multi-Drug Resistance; Multidrug-Resistant Tuberculosis; Mutation; Mutation Analysis; Mutation Detection; Mycobacterium tuberculosis; National Institute of Allergy and Infectious Disease; Patients; Performance; Pharmaceutical Preparations; Phase; Polymerase; Prevention; Price; Process; Production; Protocols documentation; Public Domains; Reaction; Reporting; Reproduction spores; Research; Research Priority; Research Support; Sampling; Sensitivity and Specificity; Side; Sputum; Surveys; System; Temperature; Testing; Tuberculosis; United States National Institutes of Health; Validation; Visit; assay development; base; biodefense; burden of illness; design; experience; human DNA; human disease; improved; instrument; instrumentation; interest; micro-total analysis system; mutant; prevent; rapid detection; rapid technique; resistance mutation; resistant strain; success; technology development; tool; tuberculosis drugs; user-friendly

DESCRIPTION (provided by applicant): Diagnosis of tuberculosis (TB) is the weakest link in global TB control and drug resistance complicates the selection of an appropriate treatment course. The most widely used diagnostic test, sputum smear microscopy, is cumbersome, insensitive, involves multiple patient visits and cannot detect multi drug or extensively drug resistant tuberculosis (MDR/XDR-TB). Sputum culture is costly and takes 4-8 weeks to detect TB and up to 16 weeks to detect MDR/XDR-TB. In mid-2011, the WHO issued a report that contains strong cautions on the use of commercial serodiagnostic test for the diagnosis of tuberculosis (WHO, 2011). There is an urgent need for a diagnostic test to simultaneously detect the presence of Mycobacterium tuberculosis (MTB) in patient samples and identify many mutations involved in MDR/XDR-TB. Our objective is to develop a multiplex ligation probe assay (MLPA)-based MTB detection and MDR/XDR mutation identification assay that can be used in moderately equipped clinical labs (the MLPA-XDR). This assay would be rapid, sensitive, specific and user-friendly, use accessible equipment and provide a simple to understand drug resistance genotype. The steps in the assay would be: (1) addition of an internal process control (such as quantified spores for extraction control) followed by standard sputum treatment and DNA isolation, (2) division of isolated DNA into MLPA reaction chambers containing prepackaged hybridization probe cocktail and process controls, (3) post- hybridization addition of ligase, (4) post-ligation PCR amplification after the addition of polymerase and two common PCR primers, and (5) imaging and image interpretation for the detection of MTB and mutations. Investigen has developed: (1) sputum processing protocols, (2) ultra-specific signature regions for MTB detection and regions of interest in drug resistance, and (3) a small set of MLPA hybridization probes that discriminate single base changes in rpoB. There is a high likelihood of success as key components have been demonstrated and the pieces simply need to be put together. This will be accomplished in three specific objectives: (1) Design MLPA LHS and RHS probes. (2) Test probe sets. (3) Test on clinical MTB and sputum samples. Investigen and collaborators have domain knowledge in MTB detection and MDR/XDR - TB mutation analysis, instrumentation/consumables prototyping, assay development, developing world product placement and global TB research agenda and treatment goals. A fuller, inexpensive MDR/XDR-TB genotyping system would greatly improve MDR/XDR-TB diagnosis and treatment. In the Phase I we intend to develop the MLPA-XDR with probes for 100 mutations. Eventually, this may be divided into a two-step approach. First an inexpensive survey of 30 or 60 common drug resistant mutations followed by an "as needed" further survey of 360 less common drug resistant mutations. Accordingly, the project is relevant to the NIH mission of reducing the burden of illness through supporting research for the improved diagnosis, prevention and cure of human diseases. The development of technologies for the easy/rapid detection of MDR/XDR-TB is a NIAID biodefense research priority. NIAID goals for TB include the development of new and improved diagnostic tools to more accurately diagnose early TB disease, help optimize therapy by identifying drug-resistant strains, and track the spread of TB in a community. If feasibility is shown (milestones in specific aims), the phase II objectives are anticipated to include; (1) instrument and consumables concepts that are geared to automation (lab on a chip), (2) inclusion of dUTP for potential amplicon contamination control, (3) determining final format and mutant panel, (4) further performance validation, (5) assay volume reduction, and (6) and assay performance and guard band optimization/characterization. PUBLIC HEALTH RELEVANCE: The feasibility of a robust method to identify multidrug resistant and extensively drug resistant tuberculosis will be explored. If successful, this can hel the global effort to control TB and prevent the spread of drug resistance.

描述（由申请人提供）：结核病的诊断是全球结核病控制中最薄弱的环节，耐药性使适当治疗方案的选择复杂化。使用最广泛的诊断检测是痰涂片镜检，这一检测方法繁琐、不敏感，涉及多次患者就诊，不能发现多药或广泛耐药结核病。痰培养费用昂贵，需要4-8周才能发现结核病，需要16周才能发现耐多药/广泛耐药结核病。2011年年中，世卫组织发布了一份报告，其中对使用商业血清诊断检测来诊断结核病提出了强烈警告（世卫组织，2011年）。迫切需要一种诊断检测方法，以同时检测患者样本中结核分枝杆菌（MTB）的存在，并确定与耐多药/广泛耐药结核病有关的许多突变。我们的目标是开发一种基于多重连接探针法（MLPA）的MTB检测和MDR/XDR突变鉴定方法，可用于中等装备的临床实验室（MLPA -XDR）。该方法具有快速、敏感、特异和用户友好性，使用方便的设备，并提供一种简单的了解耐药基因型的方法。分析的步骤如下：(1)加入内部过程对照（如定量的孢子进行提取对照），然后进行标准痰液处理和DNA分离，(2)将分离的DNA分成含有预先包装的杂交探针鸡尾酒和过程对照的MLPA反应室，(3)杂交后加入连接酶，(4)在加入聚合酶和两种常见PCR引物后进行连接后PCR扩增。(5)检测结核分枝杆菌及其突变的成像和图像解释。Investigen开发了：(1)痰液处理方案，(2)MTB检测的超特异性标记区域和耐药感兴趣的区域，以及(3)一小组区分rpoB单碱基变化的MLPA杂交探针。成功的可能性很高，因为关键组件已经展示出来，只需要将各个部分组合在一起。这将通过三个具体目标来实现：(1)设计MLPA LHS和RHS探针。(2)测试探头组。(3)临床结核分枝杆菌及痰样检测。在MTB检测和MDR/XDR - TB突变分析、仪器/耗材原型设计、检测开发、发展中国家产品植入和全球结核病研究议程和治疗目标方面，Investigen和合作者具有专业知识。一个更全面、廉价的耐多药/广泛耐药结核病基因分型系统将大大改善耐多药/广泛耐药结核病的诊断和治疗。在I期，我们打算开发具有100个突变探针的MLPA-XDR。最终，这可以分为两个步骤。首先对30或60种常见的耐药突变进行廉价调查，然后“根据需要”对360种不太常见的耐药突变进行进一步调查。因此，该项目与NIH的使命相关，即通过支持改善人类疾病的诊断、预防和治疗的研究来减轻疾病的负担。开发容易/快速检测耐多药/广泛耐药结核病的技术是NIAID生物防御研究的重点。NIAID的结核病目标包括开发新的和改进的诊断工具，以更准确地诊断早期结核病，通过识别耐药菌株帮助优化治疗，并跟踪结核病在社区中的传播。如果显示出可行性（具体目标中的里程碑），预计第二阶段的目标包括：(1)面向自动化的仪器和耗材概念（芯片上的实验室），(2)包含用于潜在扩增子污染控制的dUTP，(3)确定最终格式和突变面板，(4)进一步的性能验证，(5)测定量减少，以及(6)测定性能和保护带优化/表征。