Detection of Viral Nucleic Acids by Qβ Replicase Amplification

Abstract

The need for clinical assays of exquisite sensitivity is well established. For example, the ability to detect reliably a single infected cell present in a sample is critical in screening blood supplies for human immunodeficiency virus type 1 (HIV-1). Direct detection of nucleic acid sequences specific for the pathogen circumvents several problems associated with other assay methods. First, isolation of the organism responsible for an infection is not always possible. Second, antigen levels may be extremely low in the initial stages of infection. Finally, indirect detection is complicated by antibody titers that may be low and that may fluctuate during the course of infection. A number of methods for direct detection of low levels of particular nucleic acids have been described. Prominent among these are: the polymerase chain reaction (PCR) (Saiki et al. 1988) the transcription-based amplification system (TAS) (Kwoh et al. 1989); and the ligase chain reaction (LCR) (Backman and Wang, 1989). We have explored an alternative method of signal amplification which employs Qβ replicase to exponentially amplify probe molecules that have been bound to target nucleic acids.