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

用于诊断多重耐药结核病的 DNA 检测开关 (DDS) 探针

• 批准号: 8074033
• 负责人: DAVID A SHAFER
• 金额: $ 44.84万
• 依托单位: GENETAG TECHNOLOGY, INC.
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8074033
• 关键词: Area; Binding; Biological Assay; Centers for Disease Control and Prevention (U.S.); Cessation of life; Clinical; Clinical Management; Code; Communicable Diseases; DNA; DNA Sequence; Detection; Diagnosis; Diagnostic; Diagnostics Research; Discrimination; Doctor of Philosophy; Drug Resistant Tuberculosis; Drug resistance; Exclusion; Frequencies; G Forces; Genes; Goals; Grant; Gray unit of radiation dose; HIV; Hand; Hospitals; Incidence; Isoniazid resistance; Label; Molecular; Monitor; Morbidity - disease rate; Multi-Drug Resistance; Multidrug-Resistant Tuberculosis; Nucleic Acids; Patients; Pharmaceutical Preparations; Phase; Principal Investigator; Process; Program Development; Promoter Regions; Reader; Reporting; Resistance; Resources; Rifampicin resistance; Rifampin; Sampling; Sampling Studies; Sensitivity and Specificity; Series; Signal Transduction; Site; Synthetic Genes; System; Technology; Temperature; Testing; Time; Translating; Tuberculosis; Variant; Work; abstracting; base; blind; cost; design; improved; instrument; isoniazid; mortality; mutant; non-compliance; novel; novel diagnostics; prevent; programs; prototype; success; tool; tuberculosis drugs

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.

描述（由申请人提供）：DDS诊断探针系统用于MDR-TB首席研究员，Shafer，David A. PhD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800万例新病例和200万人死亡。近年来，抗药性结核病的发病率和传播迅速而令人震惊，多药耐药结核（MDR-TB）的频率在全球范围内增加。由于大多数新病例是由于不合规或对药物易感结核病的不当治疗而引起的，因此非常需要快速，精确的诊断，尤其是在TB和MDR-TB普遍存在的低资源环境中。这种翻译赠款正在基于PI先前发明的新的双组分误差探针技术开发一组MDR-TB的分子诊断探针，并启用了与抵抗力相关的单个碱基突变体的精确，清晰且可靠的歧视。这些探针适用于终点和阵列检测以及实时PCR（QPCR）。与QPCR的标准探针相比，这些DDS（DNA检测开关）在广泛的退火/杂交温度下提供了等效检测，它们可以选择性地检测单个基础突变体并排除在同一位点的替代变体的检测。该程序将重点介绍QPCR的一组全面的DDS探针的设计和测试，该探针可以检测RPOB基因中的主要突变体，从而赋予Rifampin（RIF）抗性以及Katg和InhA中的关键突变体位点，从而赋予ISONIAIAZID（INH）耐药性。这里报道的初步研究表明，我们已经通过重要的KATG和INHA突变体实现了绝对的单基础歧视。我们还展示了一个名为G-FORCE探针的其他基于引物的探针系统，该系统可以与内部DDS探针结合使用，从而计数突变体与扩增子频率 - 一种新的诊断能力。最初的测试将使用由主要耐药变体构建的合成基因进行。进一步的测试和盲目研究将使用来自CDC的同事提供的良好特征临床分离株和易感对照的DNA样品进行。 MDR-TB的拟议的DDS探针可以取代QPCR的标准Taqman或分子信标探针，这些探针是昂贵的，需要仔细的优化，并且容易出现假阳性。我们的DDS探测器还有望改善HAIN和INNOGENECT的线探针测定法，这些探针通常提供灰色和绝对单基抗歧视的阴影，并且经常对涂片阴性样品或HIV共感染的患者无效。我们还概述了MDR-TB的一系列潜在的二级诊断产品，这些产品将通过将这些新探针转换为简单的芯片格式，将这些新的探针转换为新的研究/诊断工具，并将其遵循并构建在此阶段I程序上，并将其转换为新的研究/诊断工具，并将这些新的探针转换为我们已经进行了原型的低成本，手持式终点读取器，并需要实时PCR仪器，从而需要实现实时PCR Inpersuments。这些短距离目标的完成应为高端研究和诊断和低资源设置中的临床管理提供宝贵的新工具。 DDS的DDS诊断探针系统用于MDR-TB主要研究者，Shafer，David A. PhD叙述：该赠款正在开发新型的诊断探针，用于实时PCR检测多药耐药性结核病（MDR-TB）。该开发计划将一个经过验证的核酸扩增平台与新的双组分误差探针技术结合在一起，称为DDS（DNA检测开关）探针，该技术是由PI发明的，并且可以高度精确，清晰，可靠的单碱基突变体在广泛的退火/杂化范围内对单个碱基突变体进行高度精确，明确且可靠的歧视。标准探针不能提供如此绝对的单基歧歧，并且需要狭窄的温度控制。该程序将重点介绍一组全面的IDD（内部DDS）探针的设计和测试，该探针可以检测RPOB基因中的主要突变位点，从而赋予KATG基因和INHA促进剂区域中赋予ISONIAIAZIDAIAZIDAIAZID（INH）的INHA启动子区域中赋予RIFAMPIN（RIF）抗性（RIF）抗性和关键突变点。 （RIF和INH是用于控制结核病的第一行药物。）该技术可以取代QPCR的标准塔克曼探针，这些QPCR的QPCR代价高昂，需要仔细优化并且容易置于假阳性，并且在Hain和Inneogenogenics的线探针测定方面改进，这些探针和Inneogenodics可以提供灰色的灰色与绝对单基歧视的阴影，并且经常与Smear Samection或HIV相关。这些诊断探针旨在用于医院和临床实验室的商业分布，用于测试和监测传染病和管理治疗。