Nucleic Acid Sequenced Based Amplification and Transcription Mediated Amplification

Nucleic Acid Sequenced Based Amplification (NASBA) and Transcription Mediated Amplification (TMA) are similar isothermal amplification techniques that proceed through RNA. Though primarily used for RNA detection, 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 included in the reaction. The RNA in the DNA-RNA hybrids is destroyed by RNase H activity (from an exogenous protein in NASBA, or by an RNase H+ RT in TMA) and dsDNA is produced by the RT. This template then gets transcribed to RNA by T7 RNAP and exponential amplification results.

TMA begins with primers targeting an RNA region of interest, one of which contains a promoter sequence for T7 RNA polymerase. The subsequent single-stranded RNA is then reverse transcribed to cDNA by an RT in the reaction. The RNase H activity of the RT degrades the RNA in the DNA-RNA hybrid as it synthesizes the complementary DNA strand. This dsDNA template is transcribed to RNA by T7 RNA polymerase, resulting in exponential amplification of the RNA target.

Detection

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, and are utilized in a range of clinical diagnostics, including those that screen for infectious diseases in the blood supply.

COVID-19

NASBA is the foundation of the two-stage COVID-19 testing strategy known as INSIGHT (isothermal NASBA sequencing–based high-throughput test), which uses a combination of NASBA and NGS technologies to combine the advantages of near-patient and centralized testing. The first stage is the NASBA reaction, which can generate rapid test results on the spot using crude saliva as sample input in 1 to 2 hours for early isolation of presymptomatic or asymptomatic patients. The stage one end product can then be sent to a central facility for pooled sequencing, allowing up to hundreds of thousands of samples to be analyzed on a single next-generation sequencer, improving the test accuracy in a highly scalable way. Details can be found in the following publication:

INSIGHT: A population-scale COVID-19 testing strategy combining point-of-care diagnosis with centralized high-throughput sequencing