Nucleic acid sequence-based amplification and transcription – mediated amplification

Also known by self-sustained sequence replications (3SR).

It is an isothermal RNA amplification method.

These isothermal assays use three enzymes:

  1. transcriptase (RT),
  2. RNAase H, and
  3. T7 DNA dependent RNA polymerase.

Here, RNA target is reverse transcribed into cDNA and then RNA copies are synthesized with the help of RNA polymerase.

It does not require thermal cycler.

NASBA based kits for are available.

Have clinical utility for the amplification of viral RNA, identification of Mycobacterium spp. antibiotic resistance, and detection of bacteria, detection and quantitation of HIV-1 RNA and CMV RNA.

DNA can also be used as a starting material.

Primers are designed to target a region of interest, but importantly, one primer includes the promoter sequence for T7 RNA polymerase at the 5′ end. This enables production of single-stranded RNA species, which are reverse transcribed to cDNA by a reverse transcriptase.

Either RNase H or a RT molecule with RNase activity degrades the RNA molecule in the RNA – DNA hybrid. (from an exogenous protein in NASBA, or by an RNase H+ RT in TMA).

The remaining cDNA molecule is replicated into double-stranded DNA molecules by the DNA polymerase activity of the polymerase (i.e., T7 bacteriophage RNA polymerase).

The RT uses a promoter that was incorporated into the cDNA engineered into the primer for the first amplification of the cDNA.

The RT then transcribes antisense RNA molecules from the cDNA molecules.

The resulting antisense RNA amplicons then continue the cycle for increased amplification of the target sequence.

NASBA reactions can be very rapid and produce a large amount of product due to the nature of T7 RNA polymerase, but much of the product will be RNA. Detection is usually accomplished using a Molecular Beacon or similar hybridization probe targeting the single-stranded product, enabling specific detection.

NASBA and TMA reactions can be extremely sensitive.