Detection of Select Agents in Single-Well Assays

单孔检测中选择试剂的检测

• 批准号: 6857104
• 负责人: David Alland
• 金额: $ 87.29万
• 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/6857104
• 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)：新出现的生物恐怖主义威胁产生了对病原菌的快速诊断分析的需要。为了最大限度地发挥效果，分析不仅必须快速、灵敏和特异，而且必须足够简单和可靠，以便在当地医疗机构和区域实验室使用。化验必须检测所有被归类为选定试剂的细菌，并应具有识别常见传染病的能力。后一项功能将鼓励人们熟悉化验方法并广泛分发，确保在发生生物恐怖袭击时使用化验方法。我们建议开发新的实时聚合酶链式反应方法，极大地扩大在一次检测中可以检测到的不同病原体的数量。这些分析方法将具有与当前一代实时聚合酶链式反应(PCR)方法相同的灵敏度和稳定性，但将具有显著扩展的区分不同病原体特定核酸序列的能力。我们的实验室率先将分子信标用作序列特异性杂交探针，用于实时聚合酶链式反应分析。在这里，我们建议开发一种新的使用分子信标的范例，这将使高度多重检测成为可能。我们将用协同作用的混合分子信标取代特定的分子信标。序列将通过检测由多个探针以特有的方式与DNA序列杂交产生的“荧光签名”来识别。从单探针鉴定到基于模式的多探针鉴定的转变将使我们能够开发仅使用少量探针或颜色的高度多元化的分析方法。我们提出了两种相关的方法。我们将创建一种“草率”的分子信标分析，它将使用六个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“颜色三重编码”格式，仅利用8个不同颜色的荧光团，就可以在同一个化验中很好地对多达56个不同的分子信标进行唯一标记。这些方法的一个显著特点是它们是基于实时聚合酶链式反应技术，这种技术已经在商业分析中得到应用。我们的具体目标是：1.开发一套“通用”的聚合酶链式反应引物，从所有选定的细菌病原体和常见的细菌病原体中扩增出物种特有的DNA序列。 2.开发能够在一次分析中很好地区分60多个不同物种特定DNA序列的聚合酶链式反应分析方法，仅利用五个不同颜色的、半特定的“马虎”分子信标。 3.发展利用“颜色三联体编码”来唯一标记多达56个不同物种特异性分子信标的聚合酶链式反应方法。