Transcription-based amplification system and detection of amplified human immunodeficiency virus type 1 with a bead-based sandwich hybridization format

Abstract

The in vitro amplification of biologically important nucleic acids has proceeded principally by a strategy of DNA replication. Polymerase chain reaction was the first such protocol to achieve this goal. In this report, a transcription-based amplification system (TAS) is described. Each cycle of the TAS is composed of two steps. The first is a cDNA synthesis step that produces one copy of a double-stranded DNA template for each copy of RNA or DNA target nucleic acid. During the course of this cDNA synthesis step, a sequence recognized by a DNA-dependent RNA polymerase is inserted into the cDNA copy of the target sequence to be amplified. The second step is the amplification of the target sequence by the transcription of the cDNA template into multiple copies of RNA. This procedure has been applied to the detection of human immunodeficiency virus type 1 (HIV-1)-infected cells. After four cycles of TAS, the amplification of the vif region of the HIV-1 RNA genome was measured to be, on the average, 38- to 47-fold per cycle, resulting in a 2-5 x 106-fold increase in the copy number of the original target sequence. This amplification by the TAS protocol allows the detection of fewer than one HIV-1-infected CEM cell in a population of 106 uninfected CEM cells. Detection of the TAS-generated RNA from HIV-1-infected cells can easily be accomplished by means of a bead-based sandwich hybridization protocol, which provides additional specificity for the identification of the amplified HIV-1-specific sequence.