How does isothermal amplification differ from PCR in coronavirus disease 2019 (COVID-19) diagnostic testing?

Updated: Jun 18, 2021
  • Author: James J Dunn, PhD, D(ABMM), MT(ASCP); more...
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The main distinction between isothermal amplification and PCR is that in the former, amplification is achieved using a constant temperature, while in the latter, cycling of temperatures is required. [40] Several methods of isothermal amplification are available, but the two most important ones for SARS-CoV-2 diagnostics are reverse transcription loop-mediated isothermal amplification (RT-LAMP) and transcript-mediated amplification (TMA). Both chemistries can generate more than 109 copies of a gene target in 1 hour. [40] Detection of the amplified target can be achieved with a nonspecific fluorescent dye, such as SYBR Green, that binds to double-stranded DNA, or a fluorescent probe that is specific to the target sequence. Some EUA assays also incorporate clustered, regularly interspaced, short palindromic repeats (CRISPR) for the detection of RNA amplified using RT-LAMP. [41, 42]

In November 2020, the FDA issued an EUA for at-home use, by prescription, of the Lucira COVID-19 All-in-One Test Kit, for persons aged 14 years or older. The kit, the first home-use COVID-19 diagnostic test, employs a self-collected nasal swab sample and RT-LAMP to detect SARS-CoV-2 RNA. A single-use device, it can provide results in 30 minutes or less. (The Lucira kit can be employed in a point-of-care setting for patients under age 14 years.) [6, 43, 44]

In March 2021, the FDA issued an EUA for the Cue COVID-19 Test for Home and Over The Counter (OTC) Use, the first at-home, nonprescription molecular COVID-19 test to be authorized by the FDA. [45]

The IDSA’s revised recommendations on COVID-19 testing suggest that rapid RT-PCR assays or standard laboratory-based NAATs be used instead of rapid isothermal NAATs when COVID-19 is suspected in symptomatic individuals. The association states that rapid isothermal NAATs appear to have lower sensitivity than the other two tests, at 81%, although the studies assessed primarily involved only one rapid isothermal NAAT, Abbott’s ID NOW. The IDSA’s recommendation did not include rapid home testing. Also, the association labeled the recommendation as conditional, with low certainty of evidence. [11, 12]

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