[Quantification of proviral FIV DNA using competitive PCR].
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 sequences. 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 distingushed by their different lengths. 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 load was studied.
Allenspach
K
K
Veterinär-Medizinisches Labor, Departement für Innere Veterinärmedizin, Universität Zürich.
Amacker
M
M
Leutenegger
C M
CM
Hottiger
M
M
Hofmann-Lehmann
R
R
Hübscher
U
U
Pistello
M
M
Lutz
H
H
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002415:Cats; D003001:Cloning, Molecular; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics; D016181:Feline Acquired Immunodeficiency Syndrome / Q000175:diagnosis*; D016089:Immunodeficiency Virus, Feline / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D051381:Rats; D012044:Regression Analysis; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004279:DNA, Viral
Die Quantifizierung von proviraler FIV-DNA mittels kompetitiver PCR.