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

描述(申请人提供)：结核病的诊断是全球结核病控制中最薄弱的一环，耐药性使选择适当的疗程变得更加复杂。最广泛使用的诊断试验是痰涂片镜检法，它笨重、不敏感、涉及多次患者探视，而且无法检测出多药或广泛耐药结核病(MDR/XDR-TB)。痰培养费用昂贵，需要4-8周来检测结核病，最多需要16周来检测耐多药/广泛耐药结核病。2011年年中，世界卫生组织发布了一份报告，对使用商业血清诊断试验诊断结核病提出了强烈警告(世卫组织，2011年)。迫切需要一种诊断测试，以同时检测患者样本中结核分枝杆菌(MTB)的存在，并确定与MDR/XDR-TB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分析(MLPA)的MTB检测和MDR/XDR突变鉴定方法，可用于中等设备的临床实验室(MLPA-XDR)。该方法具有快速、灵敏、特异性强、操作简便、使用方便等优点，并可提供一种简单易懂的耐药基因检测方法。该方法的步骤是：(1)加入内部过程对照(如用于提取控制的量化孢子)，然后进行标准的痰处理和DNA分离；(2)将分离的DNA分成MLPA反应室，其中包含预先包装的杂交探针鸡尾酒和过程对照；(3)杂交后添加连接酶；(4)在添加聚合酶和两个通用的PCR引物后进行连接后的PCR扩增；以及(5)用于检测结核分枝杆菌和突变的成像和图像解释。研究已经发展出：(1)痰处理方案，(2)用于检测结核分枝杆菌的超特异性标志区和耐药感兴趣区，以及(3)一小套区分rpoB单碱基变化的MLPA杂交探针。成功的可能性很高，因为关键组件已经得到证明，只需将这些组件组合在一起即可。这将通过三个具体目标来实现：(1)设计MLPA、LHS和RHS探头。(2)测试探针组。(3)临床结核分枝杆菌和痰标本检测。研究人员和合作者在结核杆菌检测和耐多药/广泛耐药结核突变分析、仪器/消耗品原型制作、化验开发、发展中国家的产品植入以及全球结核病研究议程和治疗目标方面拥有领域知识。一个更全面、更便宜的MDR/XDR-TB基因分型系统将极大地改进MDR/XDR-TB的诊断和治疗。在第一阶段，我们打算开发带有100个突变探针的MLPA-XDR。最终，这可能会分为两步走。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，该项目与美国国立卫生研究院的使命有关，即通过支持改善人类疾病诊断、预防和治疗的研究来减少疾病负担。开发简便/快速检测耐多药/广泛耐药结核的技术是NIAID生物防御研究的重点。NIAID针对结核病的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，通过识别耐药菌株来帮助优化治疗，以及跟踪结核病在社区中的传播。如果可行性被证明(具体目标中的里程碑)，第二阶段的目标预计将包括：(1)与自动化(芯片实验室)相适应的仪器和消耗品概念，(2)纳入dUTP用于潜在的扩增子污染控制，(3)确定最终格式和突变小组，(4)进一步的性能验证，(5)分析体积的减少，以及(6)分析性能和保护带的优化/表征。 公共卫生相关性：将探索一种可靠的方法来识别耐多药和广泛耐药结核病的可行性。如果成功，这将有助于控制结核病和防止耐药性传播的全球努力。

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.