ABI PRISM 7700 FOR TAQMAN PCR

ABI PRISM 7700 用于 TAQMAN PCR

• 批准号: 2805353
• 负责人: Jean L. Patterson
• 金额: $ 10.14万
• 依托单位: TEXAS BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-15 至 2000-04-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2805353
• 关键词: biomedical equipment; biomedical equipment purchase; nucleic acid quantitation /detection; polymerase chain reaction

Several NIH-supported research programs being conducted in the Department of Virology and Immunology at the Southwest Foundation for Biomedical Research (SFBR) would be greatly enhanced by access to an ABI PRISM 7700 sequence detector for the performance of Taqman, quantitative, real-time PCR. Taqman PCR is a method of quantitative PCR that relies on two essential attributes. The 5' nuclease PCR assay is used during PCR amplification and the fluorescence signal generated during the assay is monitored in real time. The 5' nuclease assay is similar to conventional amplification with the addition of a probe that anneals to the generated PCR product. The probe contains two dye molecules, the reporter and the quencher. The 5' nuclease activity of Taq will liberate the reporter dye from an annealed probe resulting in the generation of a fluorescent signal. This signal is measured in each cycle by fiber optics to the wells on the thermocycler (all integrated in the ABI PRISM 7700). Real time measurement in each cycle of PCR yields a quantitation over a 5-6 log range of input RNA or DNA molecules. This technology is applicable to the measurement of any nucleic acid. Within the Department of Virology and Immunology, the primary use of the instrument will be for the determination of viral load in experimentally infected animals, in human samples, and it tissue culture systems. A secondary application will be to monitor mRNA levels of cytokines and lymphocyte subset markers. The research of the department includes studies on human and similar immunodeficiency virus, simian T cell leukemia virus I, human hepatitis B, C, D and E viruses, a primate hepatitis virus, monkey herpes B virus, hemorrhagic arena- viruses, and a virus infecting Leishmania. Evaluation of the pathogenesis of these viruses in animal models, the development of vaccines and analysis of antivirals are all dependent upon the measurement of viral levels. TaqMan PCR is now the method of choice for quantitative PCR. It will result in a substantial reduction in technical time, while increasing the quality of the data. The NIH sponsored research programs of these investigators would be greatly enhanced by access to this equipment.

在西南生物医学研究基金会（SFBR）的病毒学和免疫学系进行的几个NIH支持的研究项目将通过使用ABI PRISM 7700序列检测器进行Taqman，定量，实时PCR得到极大的增强。Taqman PCR是一种依赖于两个基本属性的定量PCR方法。在PCR扩增期间使用5'核酸酶PCR测定，并且真实的监测测定期间产生的荧光信号。5'核酸酶测定类似于常规扩增，其中添加与产生的PCR产物退火的探针。探针含有两个染料分子，报告分子和猝灭分子。Taq的5'核酸酶活性将从退火的探针释放报告染料，导致荧光信号的产生。在每个循环中，通过光纤测量热循环仪上的威尔斯孔（全部集成在ABI PRISM 7700中）的信号。每个PCR循环中的真实的时间测量产生输入RNA或DNA分子的5-6个对数范围内的定量。该技术适用于任何核酸的测量。在病毒学和免疫学系，该仪器的主要用途是测定实验感染动物、人体样本及其组织培养系统中的病毒载量。第二个应用是监测细胞因子和淋巴细胞亚群标志物的mRNA水平。该部门的研究包括人类和类似的免疫缺陷病毒，猿猴T细胞白血病病毒I，人类肝炎B，C，D和E病毒，灵长类肝炎病毒，猴疱疹B病毒，出血性竞技场病毒和感染利什曼原虫的病毒的研究。在动物模型中评价这些病毒的致病性、疫苗的开发和抗病毒药物的分析都依赖于病毒水平的测量。TaqMan PCR现在是定量PCR的首选方法。这将大大减少技术时间，同时提高数据质量。这些研究人员的NIH赞助的研究项目将通过使用这种设备而得到极大的加强。