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

描述（由申请人提供）：用于MDR-TB主要研究者Shafer，大卫A.博士论文摘要：结核病（TB）是世界许多地区发病和死亡的主要原因，每年约有800万新发病例和200万例死亡。近年来，耐药结核病的发病率和传播出现了快速和惊人的增长，耐多药结核病（MDR-TB）的频率在全球范围内不断增加。由于大多数新病例是由于对药物敏感的结核病的不依从或不适当的治疗而产生的，因此迫切需要快速和精确的诊断，特别是在结核病和耐多药结核病流行的低资源环境中。这项翻译资助正在开发一套基于PI先前发明的新型双组分防错探针技术的MDR-TB分子诊断探针，该技术能够精确、明确和可靠地区分与耐药性相关的单碱基突变。这些探针适用于终点和阵列检测以及实时PCR（qPCR）。与用于qPCR的标准探针相比，这些DDS（DNA检测开关）探针在宽范围的退火/杂交温度下提供等同的检测，并且它们可以选择性地检测单碱基突变体并排除在相同位点处的替代变体的检测。该计划将专注于设计和测试一套全面的qPCR DDS探针，可以检测rpoB基因中赋予利福平（RIF）抗性的主要突变体以及katG和inhA中赋予异烟肼（INH）抗性的关键突变位点。这里报道的初步研究表明，我们已经实现了绝对的单碱基歧视与重要的katG和inhA突变体。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，可与内部DDS探针结合使用，从而计算突变体与扩增子频率-一种新的诊断能力。最初的测试将用主要耐药变异体构建的合成基因进行。进一步的测试和盲法研究将使用来自我们在CDC的同事提供的充分表征的临床分离株和泛敏感对照的DNA样本进行。所提出的用于MDR-TB的DDS探针可以取代用于qPCR的标准Taqman或分子信标探针，这些探针成本高，需要仔细优化并且容易出现假阳性。我们的DDS探针也有望改善海恩和Innogenetics的线性探针测定，其通常提供灰色阴影与绝对单碱基区分，并且通常对涂片阴性样品或HIV共感染患者无效。我们还概述了一系列潜在的耐多药结核病二级诊断产品，这些产品将遵循并建立在这一第一阶段计划的基础上，将这些新的探针转化为简单的芯片格式、新的研究/诊断工具和低成本的手持式终点读取器，我们已经制作了原型，并克服了对实时PCR仪器的需求。这些短期和长期目标的完成应该为高端研究和诊断以及低资源环境下的临床管理提供有价值的新工具。用于耐多药结核病的DDS诊断探针系统首席研究员，Shafer，大卫A.博士论文叙述：该基金正在开发用于多重耐药结核病（MDR-TB）实时PCR检测的新型诊断探针。该开发计划将成熟的核酸扩增平台与PI发明的称为DDS（DNA检测开关）探针的新型双组分防错探针技术相结合，该技术能够在广泛的退火/杂交温度范围内高度精确，清晰和可靠地区分单碱基突变体。标准探针不能提供这种绝对的单碱基区分，并且它们需要窄的温度控制。该计划将专注于设计和测试一套全面的iDDS（内部DDS）探针，可以检测rpoB基因中赋予利福平（RIF）抗性的主要突变位点，以及katG基因和inhA启动子区域中赋予异烟肼（INH）抗性的关键突变位点。(RIF和异烟肼是控制结核病的第一线药物。该技术可以取代用于qPCR的标准Taqman探针，所述标准Taqman探针昂贵、需要仔细优化并且易于出现假阳性，并且其改进了海恩和Innogenetics的线性探针测定，所述线性探针测定提供灰色阴影对比绝对单碱基区分并且通常对涂片阴性样品或HIV共感染患者无效。这些诊断探头预期用于医院和临床实验室的商业销售，用于检测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.