A Reverse Transcription Recombinase-Aided Amplification Method for Rapid and Point-of-Care Detection of SARS-CoV-2, including Variants

Abstract

The worldwide pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its emergence of variants needs rapid and point-of-care testing methods for a broad diagnosis. The regular RT-qPCR is time-consuming and limited in central laboratories, so a broad and large-scale screening requirement calls for rapid and in situ methods. In this regard, a reverse transcription recombinase-aided amplification (RT-RAA) is proposed here for the rapid and point-of-care detection of SARS-CoV-2. A set of highly conserved primers and probes targeting more than 98% of SARS-CoV-2 strains, including currently circulating variants (four variants of concerns (VOCs) and three variants of interest (VOIs)), was used in this study. With the preferred primers, the RT-RAA assay showed a 100% specificity to SARS-CoV-2 from eight other respiratory RNA viruses. Moreover, the assay here is of a high sensitivity and 0.48 copies/μL can be detected within 25 min at a constant temperature (42 °C), which can be realized on portable equipment. Furthermore, the RT-RAA assay demonstrated its high agreement for the detection of SARS-CoV-2 in clinical specimens compared with RT-qPCR. The rapid, simple and point-of-care RT-RAA method is expected to be an appealing detection tool to detect SARS-CoV-2, including variants, in clinical diagnostic applications.

Update on Rapid Diagnostics for COVID-19: A Systematic Review

“…Relative to other direct-detection methods, the RT-RAA [53], RT-LAMP [47][48][49][50][51], and MMB [54] assays reported sensitivity and specificity >90%. In studies using the LumiraDx™ SARS-CoV-2 antigen test [43][44][45], the specificity was >92%, and only in the assay performed by Krüger et al [44] the sensitivity was less than 90% (82.2%).…”

“…Lower sensitivity was also observed in samples that showed high Ct values in RT-PCR [17][18][19]24,[26][27][28][30][31][32][33][34]36,38,39,42,43,47,53], indicating a lower viral load. In SARS-CoV-2 infection, the viral load peaks in the first week after the onset of symptoms in the upper respiratory tract.…”

“…We identified 30 rapid tests that detected antigenic structures and 9 that detected SARS-CoV-2 RNA [47][48][49][50][51][52][53][54][55]. Among the assay methods, 26 were performed by lateral flow assays (considering assays with gold and fluorescence) ; 5 by Isothermal Amplification Mediated by Reverse Transcriptase (RT-LAMP) [47][48][49][50][51]; 1 by Fluorescent-Transcription probe-based real-time reverse (RT-RAA) [53]; 1 by clustered regular-spaced short palindromic repeats (CRISPR) [52]; 1 by real-time accurate portable impedimetric detection 1.0 prototype (RAPID 1.0) [55]; and 3 by microfluidic immunofluorescence (Lumi-raDx™) [43][44][45]. Furthermore, two other studies developed new diagnostic methodologies, which are the magnetic modulation biosensor (MMB) [54] and the nanozyme chemiluminescent paper test [46].…”

“…In the studies reporting direct detection, 37 reported the sensitivity and specificity of the test [47][48][49][50][51][53][54][55], 11 studies provided information about diagnostic accuracy [22,23,26,27,31,32,34,[36][37][38]54], and 2 studies did not reveal any of these parameters, mentioning only the limit of detection [46,52] (Table 1). All studies that reported sensitivity used RT-PCR variations as reference standards to determine SARS-CoV-2 infection.…”

“…Different pathogens were considered, such as Rhinovirus [26,44], Enterovirus [26], Adenovirus [44,53], Bocavirus [44], Influenza A [39,44,46,49,[53][54][55], Influenza B [39,44,[53][54][55], Metapneumovirus [44], Parainfluenza virus [44], Respiratory syncytial virus [44,54], Herpes simplex virus-2 [55], Mycoplasma pneumoniae [44], Staphylococcus aureus [44], and Streptococcus sp. [44].…”