What is NAAT?

NAAT stands for Nucleic Acid Amplification Test. It is a molecular diagnostic test targeting specific genetic sequences (nucleic acids).  In the case of SARS-CoV-2 (COVID-19), NAATs specifically target RNA (ribonucleic acid) sequences of the virus.

NAAT begins by amplifying—or replicating—the virus’ genetic material in a sample, if any is present. Through the amplification of those nucleic acids, a well-designed NAAT can detect small amounts of COVID-19 RNA in a sample, making the test highly sensitive, reliable, and less likely to return a false-negative result of an active infection.

There are several different methodologies of NAATs including PCR, NEAR, and LAMP.¹

RT (Reverse Transcriptase/Reverse Transcription)

Often you will hear these methodologies referred to as RT-PCR, RT-LAMP, or RT-NEAR. The letters “RT” stand for Reverse Transcriptase or Reverse Transcription.  RT converts RNA into DNA and is required for RNA-targeted genetic sequences.  Amplification requires a DNA sequence.

PCR (Polymerase Chain Reaction)

PCR is perhaps the most well-known NAAT methodology and is sometimes referred to as  the “gold standard” in molecular testing. In fact, new tests using other methodologies (such as LAMP) use it as their comparator method in their clinical trials.

In the case of PCR, the process requires thermocycling in which the sample is heated and cooled repeatedly. This process includes 3 stages: 1) denaturing (heating); 2) annealing (cooling); and 3) extending (heating). These stages are conducted anywhere from 20-40 times.²

Traditional PCR assays have typically used 2 primers and recognize 2 regions of target DNA. In most cases, PCR usually requires more complex equipment and additional upkeep/calibration and can be more time consuming and costly.

Examples of PCR tests include lab-based tests as well as the discontinued POC device Accula™ Dock and Covid-19 test.

NEAR (Nicking Endonuclease Amplification Reaction)

Unlike PCR, the NEAR method is an isothermal amplification reaction replicating DNA at a constant temperature using a polymerase and nicking enzyme to amplify the DNA at a temperature range of 55 °C to 59 °C. Not only is this faster than PCR, but it also has lower energy requirements.

An example of a NEAR test is the Abbott ID-Now.

LAMP (Loop-mediated Isothermal Amplification)

RT-LAMP is also an isothermal amplification method.  This newer technology is designed to detect target nucleic acid without requiring overly complicated equipment. The detection of RNA targets is accomplished by the addition of a reverse transcriptase to the LAMP reaction. In the past, LAMP systems typically used 4 to 6 primers recognizing distinct regions on the target DNA. A modern multiplex test, however, could have over 50.

One advantage of this approach is that testing can be conducted quickly in one step. The sample is mixed with very specific primers, reverse transcriptase, and DNA polymerase.  The reaction takes place under a constant temperature (60–65 °C). The fact that LAMP does not require thermocycling like PCR makes it faster and very cost effective.

RT-LAMP is well suited for all POC settings since it tends to be less expensive, rugged, and has a simpler workflow. The RT-LAMP tests may be more readily used in low to middle income communities and countries that do not have access to high tech laboratories.

The AscencioDx is a patent pending RT-LAMP platform.

Resources:

1)    Nucleic Acid Amplification Tests (NAATs) | CDC

2)    What is PCR (polymerase chain reaction)? – YourGenome