NASBA and other transcription-based amplification methods for research anddiagnostic microbiology

The qualitative and quantitative detection of RNA is important clinically. For example, genome levels can be used as a marker for replication during i nfection with an RNA virus, and the viral load can also be used to monitor antiviral therapeutic efficacy. Detection of specific mRNA may provide an a ccurate assessment of activation for persistent DNA viruses such as herpesv iruses. In bacterial infections RNA may serve as a suitable marker for bact erial viability whereas the analysis of RNA transcripts (mRNA) in eukaryote s is important for the assessment of pathogenicity or gene expression. Reve rse transcription of RNA followed by PCR (RT-PCR) has been widely employed to detect RNA species, but an often two-step reaction can be inconvenient a nd mRNA difficult to differentiate from contaminating genomic or proviral D NA. This review presents an alternative RNA amplification strategy to RT-PC R and describes the data published in studies that have used this methodolo gical approach. RNA transcription amplification (often referred to by the a cronyms NASBA, 3SR and TMA) has the advantage of being isothermal, with the reactions (including reverse transcription) occurring simultaneously in a single tube. Furthermore, the reaction is not affected by double-stranded D NA contamination so intron-nanking primers or DNase treatment are not requi red when mRNA or retroviral RNA is to be analysed. The relatively low isoth ermal temperature applied to RNA transcription amplification allows its use for insitu amplification and hybridisation without disrupting the integrit y of the cells. (C) 1999 Lippincott Williams & Wilkins.