Development and evaluation of a real-time RT-PCR assay for quantification of cell-free human immunodeficiency virus type 2 using a Brome Mosaic Virus internal control.
Quantification of cell-free virus in plasma is important for monitoring disease progression and for assessing the response to antiretroviral therapy in both human immunodeficiency type 1 and type 2 (HIV-1, HIV-2) infections. Although commercial assays suitable for HIV-1 quantification have been used for more than a decade, no commercial assays are yet available for the measurement of cell-free HIV-2. We have therefore developed a novel real-time RT-PCR assay which, unlike previously described 'in house' assays, incorporates a Brome Mosaic Virus (BMV) internal control to minimise the risk of generating false-negative or falsely low results due to unrecognised problems with viral RNA purification, cDNA synthesis or PCR amplification. The assay has a dynamic range of >5 log10, detects the clinically important HIV-2 subtypes A and B with high sensitivity and shows no cross reactivity with HIV-1. The 95% detection limit is approximately 100 HIV-2 RNA copies/ml and both the inter-assay and intra-assay variability are low (CV% at 1.8 x 10(5) copies/ml, 13.3% and 5.7%, respectively). Overall, plasma HIV-2 RNA was detected in 38% of 167 unselected HIV-2 antibody-positive samples analysed over a 2 year period. The assay described provides an ideal system for studying viral replication in HIV-2 infected patients and for monitoring antiretroviral therapy.
Centre for Virology, Department of Infection, University College London, London, UK.
R B
RB
Ferns
J A
JA
Garson
D000704:Analysis of Variance; D017795:Bromovirus / Q000235:genetics; D015658:HIV Infections / Q000821:virology; D015498:HIV-2 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D012367:RNA, Viral / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019562:Viral Load / Q000379:methods*
D012367:RNA, Viral