Rapid, easy nucleic acid-based detection of pulmonary tuberculosis

描述（申请人提供）：结核病（TB）是全球公共卫生的一个主要问题，全球近三分之一的人口（20亿人）受到感染。WHO估计，每年有1200万人发展为活动性结核病，其中200万人将死亡（WHO 50/50，2002）。目前对TB的诊断是繁琐的，并且涉及多次患者就诊和测试，包括培养痰4-8周、痰显微镜检查、基于核酸（NA）的测试和胸部X射线。迫切需要一种快速、简便的诊断测试，可以在公共卫生诊所进行，并在患者等待结果时给出诊断答案。该项目的长期目标是开发一种基于“smartDNA”的简单、快速和廉价的结核病NA诊断测试，可用于护理点（POC）。Investigen的“smartDNA技术”是一种检测TB NA的优雅而创新的方法。smartDNA是等温和比色的。目前，Investigen用于smartDNA TB检测的研究方案是：（1）常规痰液处理，（2）样品破碎，（3）通过肽核酸（PNA）-结核分枝杆菌复合体（MTC）NA的微粒捕获进行样品处理，以及（4）当第二个PNA探针与互补NA序列结合时，染料的快速催化“光激活”颜色变化。smartDNA POC TB检测试剂盒的成功开发需要六个步骤：（1）使用现成的耗材开发检测化学品和试剂;（2）改进实验室内的活化剂/读数仪;（3）使用现成的耗材开发简单的痰液处理化学品和试剂。步骤（4）至（6）将前三个步骤转化为集成的LabTop smartDNA检测系统，并配备专门设计的POC耗材。在第二阶段的建议中，我们只涉及前三个步骤。开发一个易于使用的集成系统与样品制备和NA检测是我们的长期目标。本论文的具体目标是：（1）设计MTC特异性PNA探针的特征序列;（2）优化PNA-微粒连接方法;（3）鉴定特异性捕获分离的基因组DNA中的特征NA序列的捕获PNA;（4）鉴定可以从粗细胞裂解物中捕获特征NA序列的捕获PNA;（5）鉴定有效促进“光激活”染料转化的PNA探针;（6）鉴定能够从细胞裂解物中捕获特征NA序列的捕获PNA;（7）鉴定能够从细胞裂解物中捕获特征NA序列的捕获PNA;（8）鉴定能够有效促进“光激活”染料转化的PNA探针;（9）鉴定能够从细胞裂解物中捕获特征NA序列的捕获PNA。（6）用掺入痰沉淀物中的细胞评估所开发的系统;（7）评估简化痰处理的方法;（8）改进实验室内的smartDNA激活器/读取器仪器;以及（9）用患者样本测试smartDNA系统。结核病的快速和准确诊断对于治疗结核病和减少其造成的大规模全球公共卫生威胁至关重要。在第二阶段项目期间开发的smartDNA检测将大大改善结核病诊断和治疗的状况，因此，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负担。虽然美国在检测、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结核病的传播。本项目中完成的研究将导致开发一种在检测时提供结果的护理点结核病检测;该系统可用于世界各地资源有限的环境，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部门检测，治疗和控制结核病。

DESCRIPTION (provided by applicant): With nearly one-third of the world's population (2 billion people) infected, tuberculosis (TB) is a major problem for global public health. The WHO estimates that each year 12 million people progress to active TB and 2 million of these people will die (WHO50/50, 2002). The current diagnosis for TB is cumbersome and involves multiple patient visits and tests which include culturing of sputum for 4-8 weeks, sputum microscopy, nucleic acid (NA) based tests and chest X-ray. There is an urgent need for a rapid, easy diagnostic test that could be performed at the public health clinic and give a diagnostic answer while the patient waits for the result. The long-term objective of this project is to develop a "smartDNA"-based simple, rapid and inexpensive NA diagnostic test for TB that can be used at the point-of-care (POC). Investigen's "smartDNA technology" is an elegant and innovative approach to detect TB NA. smartDNA is isothermal and colorimetric. Currently Investigen's research protocol for smartDNA TB detection is: (1) conventional sputum treatment, (2) sample disruption, (3) sample processing by peptide nucleic acid (PNA)-microparticle capture of Mycobacterium tuberculosis complex (MTC) NA, and (4) rapid catalytic 'light-activated' color change of a dye when a second PNA probe binds to complementary NA sequences. The successful development of the smartDNA POC TB detection assay requires six steps: (1) develop detection chemistry and reagents, using off the shelf consumables; (2) improve an in-lab activator/reader instrument; (3) develop simple sputum processing chemistry and reagents, using off the shelf consumables. Steps (4) to (6) translate the first three steps into an integrated LabTop smartDNA detection system with specially designed POC consumables. In this Phase II proposal, we address only the first three steps. The development of an easy to use integrated system with sample preparation and NA detection is our long term goal. The specific aims of this work are to (1) design MTC-specific PNA probes signature sequences; (2) optimize PNA-microparticle conjunction method; (3) identify capture PNAs that specifically capture signature NA sequences in isolated genomic DNA; (4) identify capture PNAs that can capture signature NA sequences from crude cell lysates; (5) identify PNA probes that efficiently promote the "light-activated" dye conversion; (6) evaluate the developed system with cells spiked into sputum sediments; (7) evaluate methods for simplification of sputum processing; (8) improve the in-lab smartDNA activator/reader instrument; and (9) test the smartDNA system with patient samples. The rapid and accurate diagnosis of TB is critical to the treatment of the disease and the reduction of the large-scale global public health threat it presents. The smartDNA assay developed during this Phase II project will greatly improve the state of TB 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. While the United States has been largely successful in detecting, treating and controlling TB, in many countries TB is a serious and growing public health issue; international travel and immigration have increased transmission of the disease. The research to be completed in this project will lead to the development of a point of care TB test that will provide a result at the time of testing; a system that can be used in resource limited settings throughout the world, in clinics and emergency rooms in the United States and potentially even at US borders - greatly aiding the public heath services in the detection, treatment and control of TB.