Quantitation of human immunodeficiency virus plasma RNA by branched DNA and reverse transcription coupled polymerase chain reaction assay methods: A critical evaluation of accuracy and reproducibility

Abstract

The present study was designed to evaluate the utility of two assays, reverse transcription coupled polymerase chain reaction (RT-PCR) and branched DNA (bDNA), to accurately and reproducibly quantitate plasma human immunodeficiency virus (HIV) RNA levels. The bDNA assay quantitated RNA transcripts, prepared from different HIV-1 subtypes (A-E), within 1.5-fold. Similarly, the bDNA assay, standardized to subtype B, was used to quantitate cultured isolates from subtypes A, C-F within 2-fold; however, the RT-PCR assay displayed a 904-fold range. Reproducibility studies demonstrated that the bDNA and RT-PCR assays could be used statistically (P<0.05) to discern less than 2- and 6.8 – 8.1-fold changes in RNA levels, respectively. This study showed that the two assay methods differ in accuracy and reproducibility. These differences need to be considered when choosing specific applications for the methods.