Detection and quantification of proviral DNA from feline immunodeficiency virus infected cats

Quantification of provirus copies is important in the context of different biological questions. The most reliable approach for DNA quantification is a PCR based on coamplification of two templates of similar length, the target sequence and the reference template, sharing the same primer recognition sequence. During the amplification, the two templates compete for the same primer set (competitive PCR, or cPCR) and consequently amplify at the same rate independently of the number of cycles. The amplified products can be distinguished by their different length. After densitometrical analysis, the proviral copy number of experimentally feline immunodeficiency virus infected cats could be calculated, since a known amount of reference template was used. The method described here proved to be very sensitive (10 copies for the competitor-DNA) and was used to quantitate the proviral load during several experiments in which the influence of periodical immunestimulations and the effect of vaccines on the virus was studied.