Ultrarapid detection of SARS-CoV-2 RNA using a reverse transcription–free exponential amplification reaction, RTF-EXPAR

Abstract

A rapid isothermal method for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, is reported. The procedure uses an unprecedented reverse transcription–free (RTF) approach for converting genomic RNA into DNA. This involves the formation of an RNA/DNA heteroduplex whose selective cleavage generates a short DNA trigger strand, which is then rapidly amplified using the exponential amplification reaction (EXPAR). Deploying the RNA-to-DNA conversion and amplification stages of the RTF-EXPAR assay in a single step results in the detection, via a fluorescence read-out, of single figure copy numbers per microliter of SARS-CoV-2 RNA in under 10 min. In direct three-way comparison studies, the assay has been found to be faster than both RT-qPCR and reverse transcription loop-mediated isothermal amplification (RT-LAMP), while being just as sensitive. The assay protocol involves the use of standard laboratory equipment and is readily adaptable for the detection of other RNA-based pathogens.

RT-qPCR Detection of SARS-CoV-2: No Need for a Dedicated Reverse Transcription Step

“…Furthermore, buffers may need to be optimised for different templates. One intriguing alternative method bypasses the RT step altogether and gives a sample-to-signal time of under 10 min [33], although its requirement for a hybridisation and nicking step relies on the identification of optimal nucleotide sequences in the target genome, which may not always be present.…”

Split T7 promoter-based isothermal transcription amplification for one-step fluorescence detection of SARS-CoV-2 and emerging variants

“…Isothermal amplification methods such as strand displacement amplification, nucleic acid sequence-based amplification, loop-mediated isothermal amplification and exponential amplification reaction have been developed to overcome the limitations of temperature control-based methods and facilitate point-of-care testing ( Li and Macdonald, 2015 ; Woo et al, 2020 ; Yuce et al, 2021 ; Carter et al, 2021 ). However, most isothermal amplification methods entail critical drawbacks: (i) it requires an initial, temperature-controlled denaturation/annealing step is required, (ii) the design of primers is complicated, and (iii) the target is limited to short RNA targets such as microRNAs with a 3′-OH group.…”

Cas12a-assisted RTF-EXPAR for accurate, rapid and simple detection of SARS-CoV-2 RNA

“…Another typical test in this category is Abbott's ID NOW COVID-19 which can obtain positive results within 5 min and negative results in 13 min based on a nicking endonuclease isothermal amplification reaction (NEAR) ( Tauh et al, 2021 ; Tu et al, 2021 ). Notably, Carter et al recently described a new isothermal method (called RTF-EXPAR) by combining exponential amplification reaction (EXPAR) with reverse transcription-free (RTF) step to convert RNA to DNA to achieve indirectly detection of the Orf1ab gene, allowing complete test within 10 min ( Carter et al, 2021 ). Despite great progress, these isothermal amplification diagnostics often suffer from problems associated with nonspecific DNA amplification, followed by high rates of false-positive diagnosis ( Chen et al, 2021b ; Feng et al, 2021 ; Reid et al, 2018 ; Schneider et al, 2019 ; Tomita et al, 2008 ).…”