Plasma SIV RNA Viral Load Determination by Real-Time Quantification of Product Generation in Reverse Transcriptase-Polymerase Chain Reaction

Abstract

Internally controlled RT-PCR methods (QC-RT-PCR) for quantification of SIV RNA are effective, but are relatively cumbersome, expensive, and time and labor intensive. For greater throughput and efficiency, we have developed a method for quantification of plasma SIV RNA levels by real-time RT-PCR using the Applied Biosystems Prism 7700 sequence detection system. This assay format allows real-time kinetic analysis of PCR product generation, providing a broad linear dynamic range and ensuring that quantification is based on analysis during the exponential phase of amplification, regardless of the input template copy number. Simultaneous amplification and analysis eliminates any requirement for handling amplified products, increasing throughput and eliminating a potential source of assay contamination. The assay we have developed for quantification of SIV RNA has a nominal threshold sensitivity of 300 copy Eq/ml of plasma, although as little as 10 copy Eq/reaction of SIV RNA template can be detected. The linear dynamic range is in excess of 5 logs. Interassay reproducibility averages 25% (coefficient of variation), based on studies of extraction and analysis of replicate aliquots of the same plasma specimens. The combination of sensitivity, precision, and broad dynamic range allows reliable quantification of viral load even during dynamic phases of SIV infection, such as through the onset and resolution of primary infection, or during treatment with antiretroviral agents. The primer-probe combinations we have developed allow quantification of SIV isolates most commonly used for experimental studies. Availability of this assay should greatly facilitate studies of basic pathogenesis and evaluation of therapeutic and prophylactic approaches in the SIV-infected macaque.

Emergence of Simian Immunodeficiency Virus-Specific Cytotoxic CD4+ T Cells and Increased Humoral Responses Correlate with Control of Rebounding Viremia in CD8-Depleted Macaques Infected with Rev-Independent Live-Attenuated Simian Immunodeficiency Virus

“…Viral loads in plasma were determined by a RT-PCR assay with a threshold of detection of 30 copies/ml (41).…”

Control of Simian/Human Immunodeficiency Virus Viremia and Disease Progression after IL-2-Augmented DNA-Modified Vaccinia Virus Ankara Nasal Vaccination in Nonhuman Primates

“…Plasma SHIV RNA levels were measured by a real-time RT-PCR assay, as described (52,53). The assay has a threshold sensitivity of 100 copy equivalents per milliliter.…”

In vivo 1H MRS of brain injury and repair during acute SIV infection in the macaque model of neuroAIDS

“…Virion associated SIV RNA in plasma was quantified by using a real-time reverse transcription-PCR assay on an Applied Biosystems (Foster City, CA) Prism 7700 sequence detection system as described previously (18,19). Results are averages of duplicate determinations.…”