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' plasma or sera using an infectious mutant virus as an internal control. The mutant virus VX-46 has a 2$-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-amplifled 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. Q u a n t i t a t i v e PCR has been used to measure the relative levels of RNA and DNA from a variety of different samples. Under ideal condit ions a direct correlat ion can be found between the a moun t of starting target material and the a m o u n t of PCR product. (1-3) However, this is often not the case for clinical samples owing to the presence of inhibitors of PCR in samples and differing efficiencies in sample recovery and kinetics of PCR34-7) Small variations in amplificat ion efficiency can change the yield of the product and make it difficult to accurately estimate the amoun t present in the starting material38) To avoid these problems, several laboratories have described the use of internal standards in PCR. (8-~4) Generally, the internal standard DNA or RNA share the same primers as the target DNA or RNA but will contain either a deletion or an insert ion so that the products obtained from the standard and targe, can be distinguished. A variat ion in the use of an internal control is the competit ive PCR procedure. <8~ In this me thod varying known amounts of the internal standards are added to equal aliquots of the sample conta in ing the u n k n o w n target sequence. The internal standard and the target sequence compete equally for primer b inding and amplif icat ion in the PCR. Variables such as the efficiency of amplif icat ion and the number of cycles will have the same effect on both templates. Equal amounts of products will be formed when the initial concentrations of the templates are equal. Experimentally, the ratio of products formed can be determined and the equivalence poin t can be calculated. This method has been successfully employed to quantify the amoun t of HIV-1 RNAs in clinical samples. (ls'16) However, this m e t h o d does not control the variat ions in RNA recovery from sample to sample. Here, we report a modif ied me thod in which an infectious HIV-1 mu tan t virus was used as the source of compet i tor RNA. Different amounts of the mu tan t virus were added to equal aliquots of the sample conta in ing an u n k n o w n a m o u n t of virus and RNA extraction and reverse transcriptase PCR (RT-PCR) were carried out in a manne r that allowed for relatively precise quant i ta ion of the a m o u n t of viral RNA present in the sample.