A rapid, POC array MDR/XDR-TB genotyping.

描述(申请人提供)：结核病的诊断是全球结核病控制的主要薄弱环节。耐药结核病化合物的诊断和治疗挑战。最广泛使用的诊断试验是痰涂片镜检法，它繁琐、不敏感，涉及多次患者探视，并且无法检测出多药或广泛耐药(MDR/XDR-TB)。痰培养费用昂贵，根据所用方法的不同，需要4-8周才能得到结果，最多需要16周才能检测出耐多药/广泛耐药结核病。因此，迫切需要一种简单、快速的诊断测试来确定结核分枝杆菌(MTB)的存在，并进行MDR/XDR基因分型，以便在患者等待期间在公共卫生诊所进行。该项目的长期目标是开发一种可在医疗点(POC)使用的结核分枝杆菌鉴定和耐多药/广泛耐药结核病基因分型检测方法。Investigen的“SmartDNAM技术”使DNA检测能够在室温下通过比色读数在均相溶液中快速进行。SmartDNA MTB鉴定分析包括：(1)常规的痰处理，(2)样品破坏，(3)通过肽核酸(PNA)-微粒捕获MTB靶标，以及(4)通过第二个(检测)PNA-DNA靶标结合染料的“光激活”颜色变化进行检测。Investigen计划将基于SmartDNA的检测扩展到MDR/XDR-TB基因分型。建议的MTB鉴定和MDR/XDR-TB基因分型方法包括(1)常规的痰治疗，(2)样品破坏，(3)PNA-微粒浓缩捕获每个感兴趣的靶区中的MTB保守序列，(4)浓缩捕获的靶标与等位基因特异性PNA阵列的元件结合，以及(5)使用SmartDNA检测所产生的PNA-DNA杂交物。POC SmartDNA MDR/XDR-TB基因分型技术的成功开发需要(1)设计用于捕获、检测和等位基因识别MDR/XDR-TB突变的探针组，以及(2)找到一种实用的方法将SmartDNA染料变色反应应用于结合在平面上的PNA-DNA杂交物。有三个具体目标。(1)展示了该系统在区分结核分枝杆菌基因组DNA中单碱基变化的有效性。(2)设计用于突变检测的探针组。(3)研制了基于PNA阵列的智能DNA读出系统。基于SmartDNA的MDR/XDR-TB基因分型分析将极大地改善结核分枝杆菌的诊断和治疗状况，因此，与NIH通过支持改善人类疾病的诊断、预防和治疗研究来减轻疾病负担的使命相关。公共卫生意义虽然美国在发现、治疗和控制结核病方面取得了很大成功，但在许多国家，结核病、耐多药结核病和广泛耐药结核病是一个严重且日益严重的公共卫生问题；国际旅行和移民增加了传播。预计将在该项目上完成的研究将导致开发一种结核病、耐多药结核病和广泛耐药结核病识别系统，该系统将在患者等待时提供结果，并可在世界各地的资源有限的环境中使用，在美国的诊所和急诊室，甚至可能在美国边境，极大地帮助公共卫生服务发现、治疗和控制结核病、耐多药结核病和广泛耐药结核病。

DESCRIPTION (provided by applicant): The diagnosis of tuberculosis (TB) is the major weak link in global TB control. Drug resistant TB compounds diagnosis and treatment challenges. 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 (MDR/XDR-TB). Sputum culture is costly and depending on the method used takes 4-8 weeks to obtain a result and up to 16 weeks to detect MDR/XDR-TB. Consequently, there is an urgent need for a simple, rapid diagnostic test to identify the presence of Mycobacterium tuberculosis (MTB) and perform MDR/XDR genotyping that could be performed at the public health clinic while the patient waits. The long-term objective of this project is to develop a MTB identification and MDR/XDR-TB genotyping test that can be used at the point-of-care (POC). Investigen's "smartDNATM technology" enables DNA detection to occur rapidly at room temperature in homogenous solution with colorimetric readout. The smartDNA MTB identification assay consists of: (1) conventional sputum treatment, (2) sample disruption, (3) MTB target capture by a peptide nucleic acid (PNA)-microparticle, and (4) detection via a `light-activated' color change of a second (detection) PNA-DNA target bound dye. Investigen plans to extend the use of smartDNA-based detection to MDR/XDR- TB genotyping. The proposed protocol for MTB identification and MDR/XDR-TB genotyping involves (1) conventional sputum treatment, (2) sample disruption, (3) PNA-microparticle enrichment capture of MTB conserved sequences in each of the target areas of interest, (4) sequence specific binding of the enrichment captured targets on elements of an allele-specific PNA array and (5) detection of the resulting PNA-DNA hybrids using smartDNA. The successful development of the POC smartDNA MDR/XDR-TB genotyping technology requires that (1) probe sets for the capture, detection and allele discrimination of mutations involved in MDR/XDR-TB be designed, and (2) a practical way is found to apply the smartDNA dye color change reaction to PNA-DNA hybrids bound to a planar surface. There are three specific aims. (1) Demonstrate utility of the conceived system to differentiate single base changes in MTB genomic DNA. (2) Design probe sets for the detection of mutations. (3) Develop smartDNA readout on a PNA array. A smartDNA-based MDR/XDR- TB genotyping assay would greatly improve the state of MTB diagnosis and treatment, and accordingly, 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. PUBLIC HEALTH RELEVANCE While the United States has been largely successful in detecting, treating and controlling TB, in many countries TB, MDR-TB and XDR-TB are a serious and growing public health issue; international travel and immigration have increased transmission. The research to be completed on this project is anticipated to lead to the development of a TB, MDR-TB and XDR-TB identification system that will provide results while the patient waits and can be used in resource constrained settings throughout the world, in clinics and emergency rooms in the United States and potentially even at US borders greatly aiding public health services in the detection, treatment and control of TB, MDR-TB and XDR-TB.