ABI PRISM 7700 FOR TAQMAN PCR

在西南生物医学研究基金会（SFBR）病毒学和免疫学部门进行的一些nih支持的研究项目将通过使用ABI PRISM 7700序列检测器来进行Taqman，定量，实时PCR的性能大大增强。Taqman PCR是一种依赖于两个基本属性的定量PCR方法。PCR扩增时采用5′核酸酶PCR法，实时监测检测过程中产生的荧光信号。5'核酸酶测定与传统扩增类似，增加了一个探针，可以对生成的PCR产物进行退火。探针包含两个染料分子，报告剂和猝灭剂。Taq的5'核酸酶活性将从退火探针中释放报告染料，从而产生荧光信号。该信号在每个循环中通过光纤测量到热循环器上的孔（全部集成在ABI PRISM 7700中）。实时测量在每个周期的PCR产生超过5-6对数范围的输入RNA或DNA分子的定量。该技术适用于任何核酸的测定。在病毒学和免疫学系内，该仪器的主要用途将是测定实验感染动物、人类样本和组织培养系统中的病毒载量。第二个应用将是监测细胞因子和淋巴细胞亚群标志物的mRNA水平。该系的研究包括人类及类似免疫缺陷病毒、猿类T细胞白血病病毒、人类乙型、丙型、丁型和戊型肝炎病毒、灵长类肝炎病毒、猴乙型疱疹病毒、出血性arena病毒和感染利什曼原虫的病毒。在动物模型中评估这些病毒的发病机制、开发疫苗和分析抗病毒药物都依赖于病毒水平的测量。TaqMan PCR是目前定量PCR的首选方法。这将大大减少技术时间，同时提高数据质量。美国国立卫生研究院赞助的这些研究人员的研究项目将大大提高这些设备的访问。

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.