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.
Highlights
The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused the worldwide COVID-19 pandemic [1,2] and has led to more than 216 million cases and4.5 million deaths globally as of 1 September 2021, according to the real-time report of the COVID-19 epidemic from WHO [3]
We developed a fluorescent-probe-based real-time reverse transcriptionRAA (RT-recombinase-aided amplification (RAA)) assay
The coverage of the sequences of the upstream primers, downstream primers and the probe was checked as 98% of approximately 360,000 genome sequences of SARS-CoV-2 with a high quality by an in-house program; the 172 sequence was highly conserved in the currently circulating variants (Figure 1), indicating that the primers/probe candidates were highly conserved and had the ability to detect variants, including these variants of concerns (VOCs) and variants of interest (VOIs)
Summary
The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused the worldwide COVID-19 pandemic [1,2] and has led to more than 216 million cases and. 4.5 million deaths globally as of 1 September 2021, according to the real-time report of the COVID-19 epidemic from WHO [3]. It has brought huge economic and social burden all over the world [4]. SARS-CoV-2 undergoes inherent gene mutation during the worldwide spread [5] and the global pandemic promotes the emergence of variants [6]. PCR methods based on highly conserved primer/probe sets targeting more SARS-CoV-2 strains are essential for the accurate detection of viral infections. Its limitations are obvious, including having no capability in medical-resource-limited areas due to its requirement of well-trained technicians and expensive laboratory instruments, and the fact that it’s time-consuming and labor-intensive [17]
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