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.
