AN INTERNALLY CONTROLLED VIRION PCR FOR THE MEASUREMENT OF HIV-1 RNA IN PLASMA

We have developed an assay to measure the HIV-1 RNA in patients' plasm a 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 t he primer-binding and major splice donor sites. To utilize this virus as an internal control, different dilutions of this virus were added t o aliquots of plasma sample to be measured, RNA was isolated and rever se-transcribed to cDNA. PCR was performed with primers selected to inc lude the sequences on either side of the insert contained in the exter nally added virus. The DNA product from the control virus is 25 bp lon ger than that from the virus present in plasma. The amount of viral RN A present in a plasma sample is calculated after the PCR-amplified pro ducts are separated by gel electrophoresis. Unlike other quantitative PCR assays, this Internally controlled virion PCR (ICVPCR) assay elimi nates errors introduced by variable recovery during the RNA purificati on step, therefore, enhancing the accuracy of the assay.