Qualitative and Quantitative Detection of Nucleic Acids of Infectious Agents by NASBA

Abstract

Since the advent of the polymerase chain reaction (PCR; Saki 1988; Mullis & Faloona 1987), a number of other nucleic acid amplification techniques have been developed. One of the most important and well developed of these new technologies is the Nucleic Acid Sequence Based Amplification (NASBA^TM method (Kievits et al 1991). NASBA^TM utilizes the coordinated activities of AMV reverse transcriptase (RT), RNase H, and T7 RNA polymerase to amplify a specific nucleic acid target. The specificity of the reaction is determined by a pair of oligonucleotide primers, which are specific for the sequence of interest. One of these primers (designated P1) is synthesized so as to include the promoter for T7 RNA polymerase as a 5’ overhang. The reaction is conducted at constant temperature (41°C) and produces a single stranded RNA product which represents a 10⁶-10⁹ amplification of the original target sequence (figure 1). Although capable of amplifying both DNA and RNA target sequences, NASBA^TM is most suitable for the amplification of RNA. Thus, NASBA^TM has become an extremely powerful technique for the detection and quantification of retroviruses (particularly HIV-1).