B. Desforges et al., COMPETITIVE REVERSE TRANSCRIPTION-POLYMERASE CHAIN-REACTION FOR QUANTIFYING MURINE AIDS VIRUS, Journal of virological methods, 62(2), 1996, pp. 161-168
Citations number
21
Categorie Soggetti
Virology,"Biochemical Research Methods","Biothechnology & Applied Migrobiology
The causative agent of murine AIDS (MAIDS) is the defective murine leu
kemia virus BM5d, that requires the replication-competent ecotropic Mu
LV (BM5e) helper virus. We developed a competitive quantitative PCR me
thod including specific internal standards to quantify, the expression
of BM5d in the spleen of infected mice and to characterize BM5d expre
ssion kinetics following experimental infection. Specimen RNA was reve
rse-transcribed and co-amplified with a competitive template containin
g a gag sequence specific for BM5d that can be discriminated from that
corresponding to wild-type cDNA by the presence of a unique restricti
on site, Bg/II. PCR products were quantified by means of densitometric
analysis after ethidium bromide staining of gels. To standardise the
RNA extraction and reverse transcription steps, the amount of defectiv
e-virus mRNA was compared to a constant copy number of murine beta acr
in mRNA. LP-BM5 production was measured in the spleen of infected mice
. Defective gag mRNA production was compared to that of the ecotropic
virus. The mRNA level of the defective virus and the titre of replicat
ive virus increased with the duration of infection, and the amount of
defective virus mRNA correlated with the titre of replicating virus.