An internally controlled virion PCR for the measurement of HIV-1 RNA in plasma

Abstract

We have developed an assay to measure the HIV-1 RNA in patients′ plasma or sera using an infectious mutant virus as an internal control. The mutant virus VX-46 has a 25-bp insert in a conserved region between the primer-binding and major splice donor sites. To utilize this virus as an internal control, different dilutions of this virus were added to aliquots of plasma sample to be measured, RNA was isolated and reverse-transcribed to cDNA. PCR was performed with primers selected to include the sequences on either side of the insert contained in the externally added virus. The DNA product from the control virus is 25 bp longer than that from the virus present in plasma. The amount of viral RNA present in a plasma sample is calculated after the PCR-amplified products are separated by gel electrophoresis. Unlike other quantitative PCR assays, this internally controlled virion PCR (ICVPCR) assay eliminates errors introduced by variable recovery during the RNA purification step, therefore, enhancing the accuracy of the assay. © 1994 by Cold Spring Harbor Laboratory Press.