Absolute Quantitation of Feline Leukemia Virus Proviral DNA and Viral RNA Loads by TaqMan® Real-time PCR and RT-PCR

Sensitive TaqMan real-time polymerase chain reaction (PCR)-based methods have been developed recently for the detection and quantitation of feline leukemia virus (FeLV) proviral DNA in infected cats. In this chapter, we outline the design and implementation of a TaqMan real-time PCR assay to quantify total FeLV proviral and viral RNA loads in infected cats. The assay is designed to amplify all three FeLV subtypes (A-C), but not FeLV-related endogenous retroviral sequences. The system is tested and optimized using proviral DNA or viral RNA from cells infected with reference strains. The sequence used to produce the standard DNA and RNA is amplified, subcloned into a vector, and sequenced. cRNA is synthesized from the linearized plasmid DNA. Standard DNA and RNA are quantified, diluted and used to determine efficiency, sensitivity, linear amplification range, and precision of the quantitative TaqMan real-time PCR assays.