Detection of Select Agents in Single-Well Assays

单孔检测中选择试剂的检测

• 批准号: 6692693
• 负责人: David Alland
• 金额: $ 47万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6692693
• 关键词: DNA primers; bacteria characteristic; biohazard detection; biotechnology; bioterrorism /chemical warfare; cooperative study; fluorescence; fluorescent dye /probe; ionophores; microorganism classification; nucleic acid amplification techniques; nucleic acid hybridization; nucleic acid probes; nucleic acid quantitation /detection; nucleic acid sequence; polymerase chain reaction; rapid diagnosis; technology /technique development

DESCRIPTION (provided by applicant): The emerging threat of bioterrorism has created a need for rapid diagnostic assays of pathogenic bacteria. To be maximally effective, assays must not only be rapid, sensitive, and specific, but also sufficiently simple and robust to be used in local healthcare settings, as well as in regional laboratories. Assays must detect all bacteria that are classified as select agents, and should also have the capacity to identify common infectious diseases. This latter feature will encourage both familiarity with the assays and their widespread distribution, assuring their availability if a bioterrorist attack occurs. We propose the development of novel approaches to real-time PCR that vastly expand the number of different pathogens that can be detected in a single assay well. These assays will have the same sensitivity and robustness as the current generation of real-time polymerase chain reaction (PCR) methods, but will have a dramatically expanded ability to distinguish different pathogen-specific nucleic acid sequences. Our laboratories have pioneered molecular beacons as sequence-specific hybridization probes for use in real-time PCR assays. Here, we propose the development of a new paradigm for the use of molecular beacons that will enable highly multiplexed detection. We will replace specific molecular beacons with mixtures of molecular beacons that act in concert. Sequences will be identified by detecting "fluorescence signatures" generated by multiple probes that hybridize to DNA sequences in characteristic fashions. The switch from single-probe identification to multi-probe, pattern-based identification will enable us to develop highly multiplexed assays using only a small number of probes or colors. We propose two related approaches. We will create a "sloppy" molecular beacon assay that will use six differently colored molecular beacons to distinguish among 60 or more different target sequences. We will also develop a "color-triplet coding" format that will enable unique labeling of as many as 56 different molecular beacons in the same assay well, utilizing only eight differently colored fluorophores. A distinguishing feature of these approaches is that they are based on real-time PCR technology, which has already been reduced to practice in commercial assays. Our specific aims: 1. To develop sets of "universal" PCR primers that amplify species-specific DNA sequences from all select bacterial agents and common bacterial pathogens. 2. To develop PCR assays that are able to distinguish over 60 different species-specific DNA sequences in a single assay well, utilizing only five differently colored, semi-specific "sloppy" molecular beacons. 3. To develop PCR assays that utilize "color-triplet coding" to uniquely label as many as 56 different species-specific molecular beacons.

描述（由申请人提供）：生物恐怖主义的新威胁产生了对病原菌快速诊断测定的需求。为了获得最大的效果，检测不仅必须快速、灵敏和特异，而且还必须足够简单和稳健，以便在当地医疗机构以及区域实验室中使用。检测必须检测所有被归类为特定病原体的细菌，并且还应该具有识别常见传染病的能力。后一个功能将鼓励人们熟悉这些检测方法及其广泛分布，确保在发生生物恐怖袭击时它们的可用性。我们建议开发实时 PCR 的新方法，从而大大增加在单个检测孔中可以检测到的不同病原体的数量。这些检测方法将具有与当前一代实时聚合酶链反应（PCR）方法相同的灵敏度和稳健性，但区分不同病原体特异性核酸序列的能力将显着增强。我们的实验室率先将分子信标作为序列特异性杂交探针用于实时 PCR 检测。在这里，我们建议开发一种使用分子信标的新范例，以实现高度多重检测。我们将用协同作用的分子信标混合物取代特定的分子信标。通过检测多个探针产生的“荧光特征”来识别序列，这些探针以特征方式与 DNA 序列杂交。从单探针识别到多探针、基于模式的识别的转变将使我们能够仅使用少量探针或颜色来开发高度多重检测。我们提出了两种相关的方法。我们将创建一个“草率”的分子信标测定，它将使用六种不同颜色的分子信标来区分 60 个或更多不同的目标序列。我们还将开发一种“颜色三联体编码”格式，仅利用八种不同颜色的荧光团，即可在同一测定孔中对多达 56 个不同的分子信标进行独特标记。这些方法的一个显着特征是它们基于实时 PCR 技术，该技术已经在商业化验中得到应用。我们的具体目标： 1. 开发一套“通用”PCR 引物，用于扩增所有选定细菌病原体和常见细菌病原体的物种特异性 DNA 序列。 2. 开发能够在单个测定孔中区分 60 多种不同物种特异性 DNA 序列的 PCR 测定方法，仅使用五种不同颜色的半特异性“草率”分子信标。 3. 开发利用“颜色三联体编码”来独特标记多达 56 个不同物种特异性分子信标的 PCR 检测方法。