NUCLEIC ACID SEQUENCE‐BASED AMPLIFICATION (NASBA) IN MOLECULAR BACTERIOLOGY: A PROCEDURAL GUIDE

Abstract

ABSTRACT 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. PRACTICAL APPLICATIONSAlthough not a truly new laboratory method for nucleic acid analysis, NASBA has gained a popular following in recent years in the scientific literature. A diverse array of life science applications for NASBA have developed due to the advent of real‐time detection methods. The field of molecular bacteriology has witnessed a plethora of reports on the use of NASBA in fields ranging from molecular and clinical diagnostics to food microbiology and environmental monitoring. NASBA offers very high speed and sample throughput capabilities, with the sensitivity of real‐time detection chemistries, without necessarily having to confirm or validate signal/amplicon identity. What follows is both a background on the uses of NASBA in bacteriology, as well as a procedural guide on preparing target RNA for subsequent RNA analysis by NASBA. This paper is written from a benchtop pedagogical standpoint and so makes a suitable reference for those learning or teaching NASBA to others.