Competitive polymerase chain reaction for quantitating feline immunodeficiency virus load in infected cat tissues.
To quantitate FIV provirus copy numbers present in tissue of infected cats, we have applied a competitive polymerase chain reaction (cPCR) recently described for HIV. The method consists in coamplifying a fixed amount of the DNA to be examined with graded copy numbers of a DNA competitor incorporating a short deletion and bearing the same primer recognition sequences. These conditions ensure almost identical thermodynamic and amplification efficiency for both template species but permit a prompt recognition of the two amplification products by gel electrophoresis. Since the amounts of the two amplicons are dependent on relative initial template concentrations, the number of FIV genomes in the sample can be calculated by densitometric analysis of the electrophoretic bands. After validation, the method has been applied to study the provirus loads in the tissues of cats infected with the Pisa-M2 isolate of FIV.
Department of Biomedicine, University of Pisa, Italy.
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M
Pistello
S
S
Menzo
M
M
Giorgi
L
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Da Prato
G
G
Cammarota
M
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Clementi
M
M
Bendinelli
D000818:Animals; D001483:Base Sequence; D001853:Bone Marrow / Q000737:chemistry*; D002415:Cats; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics*; D003720:Densitometry; D016181:Feline Acquired Immunodeficiency Syndrome / Q000821:virology; D016089:Immunodeficiency Virus, Feline / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*
D004279:DNA, Viral