Nucleic acid sequence-based amplification (NASBA) is an isothermal-based method of RNA amplification (Davey and Malek, 1989). Using this method, RNA is amplified by the action of an enzyme cocktail that includes AMV Reverse Transcriptase, T7 RNA polymerase and RNAse H at a fixed temperature (41°C; Figure 1). By pairing this technique with the ability to monitor the fluorescence signal produced from Molecular Beacon probes (Figure 2) in real time as they hybridize to the amplicon, it is possible to perform real time analysis of samples and obtain data in a matter of minutes. By coupling these technologies with a hand-held detection system, this method becomes a deployable monitoring device for onboard and remote sensing purposes.
Figure 1. NASBA amplification pathway. Target ssRNA
(in this case, Noroviral genome) binds to Primer 1. An RNA/DNA hybrid
is formed by the action of reverse transcriptase. RNaseH then degrades
the RNA component of the hybrid and reverse transcriptase using Primer
2 makes a cDNA of the target region. Because Primer 1 contains a T7
RNA polymerase promoter, many copies of the target RNA are made. NASBA
reagents are available from Biomerieux under the product name Nuclisens
http://www.biomerieux-usa.com/clinical/nucleicacid/index.htm
Figure 2. Molecular Beacon probe in the unbound hairpin conformation (upper figure) and in the bound, fluorescent conformation. Amplification of the target sequence can be monitored by fluorescence measurements made every minute.
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