G. Cammarota et al., QUANTITATION OF FELINE IMMUNODEFICIENCY PROVIRUSES IN DOUBLY INFECTEDCATS USING COMPETITIVE PCR AND A FLUORESCENCE-BASED RFLP, Journal of virological methods, 62(1), 1996, pp. 21-31
Citations number
14
Categorie Soggetti
Virology,"Biochemical Research Methods","Biothechnology & Applied Migrobiology
A nested polymerase chain reaction assay, which amplifies a region of
the gag gene, was developed for the direct detection of feline immunod
eficiency virus (FIV) DNA sequences in the blood of infected cats. Thi
s method detects as few as ten copies of a plasmid containing the whol
e genome of the FIV-Pet isolate on agarose gel. To distinguish two FIV
isolates in double infected cats: we devised an RFLP analysis on PCR
amplified products exploiting sequence differences in the gag gene of
the two strains. To quantitate the two strains, a fluorescent inner-se
nse primer was used in the second amplification step. Amplicons were s
ubsequently digested, heat-denatured and loaded on a polyacrylamide ge
l in an automated DNA sequencer. The proportion of the two isolates wa
s determined using the laser-excited fluorescence of labelled strain s
pecific fragments. These data were used to extrapolate the numbers of
proviral genomes from the total viral load as estimated by using a com
petitive PCR assay, These sensitive and specific assays complement vir
ological detection of FIV and enable superinfection studies to be eval
uated; a prerequisite for the testing of live attenuated immunodeficie
ncy virus vaccines.