DETECTION AND QUANTIFICATION OF PROVIRAL DNA FROM FELINE IMMUNODEFICIENCY VIRUS-INFECTED CATS

Quantification of provirus copies is important in the context of diffe rent biological questions. The most reliable approach for DNA quantifi cation is a PCR based on coamplification of two templates of similar l ength, the target sequence and the reference template, sharing the sam e primer recognition sequence. During the amplification, the two templ ates compete for the same primer set (competitive PCR, or cPCR) and co nsequently amplify at the same rate independently of the number of cyc les. The amplified products can be distinguished by their different le ngth. After densitometrical analysis, the proviral copy number of expe rimentally feline immunodeficiency virus infected cats could be calcul ated, since a known amount of reference template was used. The method described here proved to be very sensitive (10 copies for the competit or-DNA) and was used to quantitate the proviral load during several ex periments in which the influence of periodical immunestimulations and the effect of vaccines on the virus was studied.