Quantitative RT-PCR assay for SIV RNA using automated real time quantitation of PCR product generation

To circumvent problems inherent in the use of endpoint PCR for quantitative applications, we have used internally controlled PCR methods (QC-PCR) for quantitation of HIV and SIV RNA. While sensitive, accurate, and reproducible, such approaches are labor intensive. We developed a quantitative RT-PCR assay for SIV RNA, using the ABI Prism 7700 Sequence Detection System and TaqMan chemistry in which a fluorescent signal is generated proportional to the number of rounds of Taq-mediated copying of specific target template sequences. Multiple times during each amplification cycle, laser optics excite each of 96 ongoing amplification reactions. The resulting emitted fluorescent signal, reflecting PCR product generation, is quantitated, permitting real time tracking of PCR product generation and kinetic analysis, based on the amplification cycle at which an above threshold level of fluorescence is first detected. A standard curve of such "threshold cycle" values vs. known input control template copy numbers allows template copy number in unknown samples to be calculated by interpolation of measured threshold cycle values; internal controls can also be incorporated. Real time kinetic analysis provides a large dynamic range and in contrast to endpoint approaches ensures that all determinations are performed during the early exponential phase of amplification, when PCR fidelity and accuracy are greatest. The two enzyme RTPCR assay we have developed can detect as few as 10-20 nominal copies/reaction of SIV gag template, while providing more than 5 logs of linear dynamic range (typical R(2) greater than 0.98). Reproducibility is comparable to QC-PCR, using quantitated in vitro transcripts (interassay C.V. 12-20%) or plasma from SIV infected macaques (interassay C.V. 19-27%) as template. The assay format is rapid and convenient, does not require any manipulation of amplified products and permits quantitation of in vivo viral load for both SIVmac and SIVsm isolates of interest.

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