Nucleic acid sequence-based amplification (NASBA) in molecular bacteriology: A procedural guide

As a means of assessing gene expression and RNA structure/function, nucleic acid sequence-based amplification (NASBA) involves an isothermic series of reactions using avian myeloblastosis virus reverse transcriptase (RT), RNase H, T7 RNA polymerase, transcript-specific primers and associated cofactors to amplify large amounts of target RNA. NASBA offers greater speed, sensitivity and versatility compared to other RNA analyses (e.g., RT-PCR, RNase protection assays and Northern blotting). NASBA allows for target RNA detection by real-time chemistries, such as SYBR dyes or molecular beacon probes, an advantage in minimizing contamination considering the closed-tube format of this approach. NASBA has been utilized in diagnostic bacteriology for clinical, environmental and food applications. NASBA kits are currently available for RNA amplification and analyses, a market primarily aimed at the clinical microbiology arena. This article will overview the background and application of NASBA, followed by protocols commonly used for bacterial diagnostics and genomic studies in various experiments.