NASBA® (Nucleic Acid Sequence Based Amplification) is an isothermal nucleic acid amplification technology allowing the amplification of RNA or DNA targets through a transcription process, after insertion of a T7 promotor. NASBA® technology is based on the concerted action of three enzymes (fig. 1):
- AMV Reverse Transcriptase: for cDNA synthesis
- RNase H: for degradation of the RNA in the heteroduplex RNA-DNA
- T7 RNA polymerase: for synthesis of RNA from the T7 promoter
Figure 1: NASBA reaction
Figure 2: kinetic of beacon hybridization | Specific primers are used, one of them carrying the T7 promoter. RNA and DNA targets are greatly amplified up to one billion fold in within 60 to 90 minutes reaction time. The amplification reaction is isothermal, proceeds at 41°C,and results in single stranded RNA molecule synthesis. Isothermal NASBA allows amplification runs, in parallel, consisting of multiple targets. The detection is performed with specific probes in a stem-loop structure called beacons (fig. 2), carrying a fluorescent molecule and a quencher at their extremities. The loop sequence of the beacon is specific and complementary to the nucleic acid sequence targeted. The beacon then binds to the target and when it is opened, the quencher is distant from the fluorescent molecule and consequently allows the emission of fluorescence (fig.2). |
The formation of the newly generated RNA molecules is therefore determined in real time by continuous monitoring of fluorescence in a dedicated reader: the NucliSENS EasyQ® System (fig.3).
Figure 3: NucliSENS EasyQ® platform
The detection of amplification products generated during an amplification reaction is monitored in “real time” and takes place in a single closed tube. bioMérieux has combined two state-of-the-art technologies, NASBA® amplification and Molecular Beacon based probe detection, for a new real time diagnostic test called NucliSENS EasyQ®. The NucliSENS EasyQ® test format has been designed to significantly reduce contamination risks and technical hands-on-time and to provide rapid results, thereby meeting the needs of the routine clinical laboratory for research applications and nucleic acid “home brew” testing.
NASBA® and NucliSENS EasyQ® are registered trademarks of bioMérieux (Marcy L’Etoile, France)