Influence of preassay and sequence variations on viral load determination by a multiplex real-time reverse transcriptase-polymerase chain reaction for feline immunodeficiency virus

Determination of retroviral load is an important tool in the investigation of the success of therapeutic or vaccination trials in patients infected wi th lentiviruses such as HIV, or with their simian (SIV) or feline (FIV) cou nterparts. We have developed an one-tube quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay based on the ABI Prism 7700 Sequence Detecti on System (TaqMan) to quantify the viral load of FIV-infected cats. Two dif ferent primer/probe systems were designed to detect a broad range of clade A FIV isolates. Both systems are characterized by excellent reproducibility , high sensitivity, and a wide range of quantification. As a consequence of this improved precision in the quantitative RT-PCR, preassay variations ha ve greater impact on the accuracy of the viral load estimation. To compensa te for these variations, we improved the assay and developed a multiplex re al-time RT-PCR, which allows simultaneous calculation of the viral copy num ber and the individual recovery rate in an one-tube reaction. This enables the rapid and accurate calculation of copy number independent of preassay v ariations. In further studies, two additional real-time RT-PCR assays were designed and used to investigate the influence of sequence variations in th e binding regions for either the primers or probe. Sequence mismatches in t his region had a significant effect (up to 4 logarithmic decades) on reacti on efficiency. In view of the inherent variability of retroviral sequences, these results underline the necessity to check reaction efficiencies befor e determining viral load.