Development and comparison of strain specific gag and pol real-time PCR assays for the detection of Visna/maedi virus.
The aim of this study was the development of gag and pol dual labelled probe real-time PCR and RT PCR assays to quantify the proviral load and the transcripts of the British Visna/maedi virus EV1 strain. Primers and probes were chosen based on the consensus sequences of gag and pol clones representative of EV1 genetic variants. Both PCRs had a detection limit of 3 copies of target gene, with a linearity over 6 orders of magnitude. The performances of the two PCRs in vivo were evaluated and compared on a panel of DNAs extracted from blood of sheep infected experimentally with EV1. The pol assay detected in most cases lower numbers of viral molecules than gag assay, yielding some false negative results. The gag real-time RT PCR had a detection limit of 100 RNA molecules with a linearity over 5 orders of magnitude. This did not result in a lower performance of the RT PCR compared to the PCR in cells permissive for virus replication, which contain higher numbers of viral transcripts than proviral genomes. The real-time assays developed in this study, particularly the gag assay, provide a sensitive tool which can be used to quantify the viral load in experimental infections.
Carrozza
Maria Luisa
ML
Scuola Normale Superiore, 56126 Pisa, Italy. carrozza@vet.unipi.it
Mazzei
Maurizio
M
Bandecchi
Patrizia
P
Fraisier
Christophe
C
Pérez
Marta
M
Suzan-Monti
Marie
M
de Andrés
Damian
D
Amorena
Beatriz
B
Rosati
Sergio
S
Andrésdottir
Valgerdur
V
Lujan
Lluis
L
Pepin
Michel
M
Blacklaws
Barbara
B
Tolari
Francesco
F
Harkiss
Gordon D
GD
D000818:Animals; D001483:Base Sequence; D015750:Genes, gag; D015751:Genes, pol; D008969:Molecular Sequence Data; D011021:Pneumonia, Progressive Interstitial, of Sheep / Q000175:diagnosis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D012756:Sheep; D019562:Viral Load / Q000379:methods*; D016182:Visna / Q000175:diagnosis*; D014790:Visna-maedi virus / Q000235:genetics / Q000302:isolation & purification*