DNA Detection Switch (DDS) probes for diagnosis of multi-drug resistant tuberculo

描述(申请人提供)：耐多药结核病的DDS诊断探针系统，首席研究员，Shafer，David A.博士摘要：结核病(TB)是世界许多地区发病率和死亡率的主要原因，每年约有800万新病例和200万死亡。近年来，耐药结核病的发病率和传播速度迅速上升，在全球范围内，耐多药结核病(MDR-TB)的发病率也在不断增加。由于大多数新病例是由不遵守或不适当的药物敏感结核病治疗引起的，迫切需要快速和准确的诊断，特别是在结核病和耐多药结核病流行的低资源环境中。这项翻译赠款正在开发一套针对耐多药结核病的分子诊断探针，该探针基于PI以前发明的新的双组分防错探针技术，能够精确、明确和可靠地区分与耐药性相关的单碱基突变。这些探针适用于末端和阵列检测以及实时荧光定量聚合酶链式反应(QPCR)。与qPCR的标准探针相比，这些DDS(DNA检测开关)探针在广泛的退火/杂交温度范围内提供了同等的检测能力，并且它们可以选择性地检测单个碱基突变，并排除同一位置的其他变异检测。该计划将专注于设计和测试一套全面的用于qPCR的DDS探针，该探针可以检测rpoB基因中导致利福平(RIF)耐药性的主要突变，以及katG和inha中导致异烟肼(INH)耐药性的关键突变位点。这里报告的初步研究表明，我们已经与重要的katG和inha突变体实现了绝对的单碱基区分。我们还展示了一种额外的基于引物的探针系统，称为G-Force探针，它可以与内部DDS探针一起使用，从而计算突变与扩增子频率-这是一种新的诊断能力。初步测试将使用用主要耐药变异构建的合成基因进行。我们将使用疾控中心同事提供的具有良好特征的临床分离株和泛敏感对照的DNA样本进行进一步的测试和盲法研究。建议的用于耐多药结核病的DDS探针可以取代用于qPCR的标准Taqman或分子信标探针，后者成本高昂，需要仔细优化，并且容易出现假阳性。我们的DDS探针还有望改进Hain和Innogenology的线性探针分析，后者通常提供灰色阴影与绝对单碱基区分，并且对涂片阴性样本或HIV联合感染患者经常无效。我们还概述了一系列潜在的耐多药结核病辅助诊断产品，这些产品将在这一第一阶段计划的基础上，通过将这些新探头转化为简单的芯片格式、新的研究/诊断工具以及低成本的手持终端读取器来实现，我们已经制作了这种读取器的原型，并克服了对实时聚合酶链式反应仪器的需求。这些短期和长期目标的完成将为高端研究和诊断以及在低资源环境下的临床管理提供有价值的新工具。耐多药结核病(MDR-TB)的DDS诊断探针系统首席研究员谢弗博士描述：这笔赠款正在开发新的诊断探针，用于对耐多药结核病(MDR-TB)进行实时聚合酶链式反应检测。该开发计划将成熟的核酸扩增平台与PI发明的名为DDS(DNA检测开关)探针的新型双组分防错探针技术相结合，能够在广泛的退火/杂交温度范围内对单碱基突变进行高精度、清晰和可靠的区分。标准探针不提供这种绝对的单碱基分辨，它们需要较窄的温度控制。该计划将专注于设计和测试一套全面的IDDS(内部DDS)探针，这些探针可以检测rpoB基因中导致利福平(RIF)耐药性的主要突变位点，以及katG基因和inha启动子区域中导致异烟肼(INH)耐药性的关键突变位点。(RIF和INH是结核病控制的一线药物。)这项技术可以取代用于qPCR的标准Taqman探针，后者昂贵、需要仔细优化且容易出现假阳性，它改进了Hain和Innogenology的线性探针分析，后者提供灰色阴影与绝对单碱基区分，并且对涂片阴性样本或HIV联合感染患者经常无效。这些诊断探头旨在用于医院和临床实验室的商业分发，用于测试和监测传染病以及管理治疗。

DESCRIPTION (provided by applicant): DDS diagnostic probe systems for MDR-TB Principal Investigator, Shafer, David A. PhD Abstract: Tuberculosis (TB) is a primary cause of morbidity and mortality in many areas of the world with about 8 million new cases and 2 million deaths per year. In recent years, there has been a rapid and alarming increase in the incidence and spread of drug resistant TB and the frequency of multi-drug resistant TB (MDR-TB) is increasing worldwide. With most new cases arising from non-compliance or inappropriate treatment of drug susceptible TB, there is a serious need for rapid and precise diagnosis, particularly in the low-resource settings where TB and MDR-TB are prevalent. This translational grant is developing a set of molecular diagnostic probes for MDR-TB based upon new dual-component error-preventing probe technologies that were invented previously by the PI and that enable precise, clear-cut and reliable discrimination of single base mutants associated with resistance. These probes are suitable for end-point and array detection as well as real-time PCR (qPCR). Compared to standard probes for qPCR, these DDS (DNA Detection Switch) probes provide equivalent detection over a wide range of annealing/hybridization temperatures and they can selectively detect a single base mutant and exclude detection of alternate variants at the same site. The program will focus on the design and testing of a comprehensive set of such DDS probes for qPCR that can detect the primary mutants in the rpoB gene that confer Rifampin (RIF) resistance and the key mutant sites in katG and inhA that confer Isoniazid (INH) resistance. Preliminary studies reported here demonstrate we have already achieved absolute single base discrimination with important katG and inhA mutants. We have also demonstrated an additional primer-based probe system called G-Force probes that can be used in conjunction with the internal DDS probes to thereby count the mutant vs. amplicon frequency - a new diagnostic capacity. Initial testing will be done with synthetic genes constructed with the major drug resistant variants. Further testing and blind studies will be conducted with DNA samples from well-characterized clinical isolates and pan- susceptible controls that are being provided by our colleagues at the CDC. The proposed DDS probes for MDR-TB can supplant standard Taqman or Molecular Beacon probes for qPCR that are costly, require careful optimization and are prone to false positives. Our DDS probes are also expected to improve upon the line probe assays of Hain and Innogenetics that typically provide shades of gray vs. absolute single base discrimination and that are frequently ineffective with smear negative samples or HIV co-infected patients. We have also outlined a series of potential secondary diagnostic products for MDR-TB that will follow and build on this Phase I program by translating these new probes to a simple chip format, to new research/diagnostic tools, and to a low-cost, hand- held end-point reader that we have already prototyped and that overcomes the need for real-time PCR instruments. The completion of these short and long range goals should provide valuable new tools for high end research and diagnostics and for clinical management in low resource settings. DDS diagnostic probe systems for MDR-TB Principal Investigator, Shafer, David A. PhD Narrative: This grant is developing novel diagnostic probes for real-time PCR detection of multi-drug resistant tuberculosis (MDR-TB). The development program combines a proven nucleic acid amplification platform with new dual-component error-preventing probe technologies called DDS (DNA detection switch) probes that were invented by the PI and that enable highly precise, clear-cut and reliable discrimination of single base mutants over a wide range of annealing/hybridization temperatures. Standard probes do not provide such absolute single base discrimination and they require narrow temperature control. The program will focus on the design and testing of a comprehensive set of iDDS (internal DDS) probes that can detect the primary mutant sites in the rpoB gene that confer Rifampin (RIF) resistance and key mutant sites in the katG gene and the inhA promoter region that confer resistance to Isoniazid (INH). (RIF and INH are the first line drugs for TB control.) This technology can supplant standard Taqman probes for qPCR which are costly, require careful optimization and are prone to false positives, and it improves upon the line probe assays of Hain and Innogenetics that provide shades of gray vs. absolute single base discrimination and that are frequently ineffective with smear negative samples or HIV co-infected patients. These diagnostic probes are intended for commercial distribution in hospital and clinical labs for testing and monitoring infectious disease and for managing treatment.