Advances in molecular biology have led to the development of more sensitive, more specific and more rapid testing procedures based on nucleic acid analysis. The introduction of nucleic acid amplification techniques, in particular, has had a major impact on the way in which nucleic acid analysis is performed. These techniques enable the amplification of small quantities of specific nucleic acid target sequences to concentrations well above the detection limit of many conventional detection methods.
Nucleic acid amplification technologies have become powerful investigation tools for scientists from a wide range of disciplines in the research field. The NASBA® method is a versatile technique particularly adapted for applications requiring specific amplification of nucleic acid sequences of viral genomes, genomes of other infectious or pathogenic agents or certain cellular mRNA.
NASBA® (Nucleic Acid Sequence Based Amplification) is an isothermal nucleic acid amplification technology allowing the amplification of RNA or DNA targets (with a slight modification in the protocol) 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 promotor
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. 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). For a typical assay, the reaction time for real-time amplification and detection is just one hour.
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 testing.
Figure 3: NucliSENS EasyQ® platform