Reverse Transcription Recombinase-Aided Amplification Assay With Lateral Flow Dipstick Assay for Rapid Detection of 2019 Novel Coronavirus

Abstract

BackgroundThe emerging Coronavirus Disease-2019 (COVID-19) has challenged the public health globally. With the increasing requirement of detection for SARS-CoV-2 outside of the laboratory setting, a rapid and precise Point of Care Test (POCT) is urgently needed.MethodsTargeting the nucleocapsid (N) gene of SARS-CoV-2, specific primers, and probes for reverse transcription recombinase-aided amplification coupled with lateral flow dipstick (RT-RAA/LFD) platform were designed. For specificity evaluation, it was tested with human coronaviruses, human influenza A virus, influenza B viruses, respiratory syncytial virus, and hepatitis B virus, respectively. For sensitivity assay, it was estimated by templates of recombinant plasmid and pseudovirus of SARS-CoV-2 RNA. For clinical assessment, 100 clinical samples (13 positive and 87 negatives for SARS-CoV-2) were tested via quantitative reverse transcription PCR (RT-qPCR) and RT-RAA/LFD, respectively.ResultsThe limit of detection was 1 copies/μl in RT-RAA/LFD assay, which could be conducted within 30 min at 39°C, without any cross-reaction with other human coronaviruses and clinical respiratory pathogens. Compared with RT-qPCR, the established POCT assay offered 100% specificity and 100% sensitivity in the detection of clinical samples.ConclusionThis work provides a convenient POCT tool for rapid screening, diagnosis, and monitoring of suspected patients in SARS-CoV-2 endemic areas.

Recombinase-Aided Amplification Coupled with Lateral Flow Dipstick for Efficient and Accurate Detection of Porcine Parvovirus

“…Nowadays, the applications of RAA in the detection of viruses, bacteria, and parasites are being widely explored. For example, Zheng et al [ 24 ] established an RT-RAA-LFD assay for the rapid diagnosis and endemic monitoring of Coronavirus Disease-2019. Zhang et al [ 25 ] developed a real-time fluorescence RAA detection to monitor foodborne salmonella contamination.…”

Assessment of the Diagnostic Ability of Four Detection Methods Using Three Sample Types of COVID-19 Patients

“…Furthermore, minimal requirements for equipment and resources make it especially suitable for on-site inspection in the underdeveloped areas, which has broad application prospects ( Li et al., 2020 ). It was reported that the established POCT assay-based RAA offered 100% specificity and 100% sensitivity in the detection of clinical respiratory tract samples from COVID-19 patients when compared with RT-PCR ( Xue et al., 2020 ; Zheng et al., 2021 ). Additionally, a multiple-center clinical evaluation of RT-RAA kit using respiratory tract samples (throat swabs, sputum, nasopharyngeal swabs, nasal swabs and bronchoalveolar lavage fluid) and non-respiratory samples (stool and whole blood) show that the total coincidence rate was 97.78% and the kappa value 0.952 (p < 0.05) compared to the commercial RT-PCR kits ( Wang J. et al., 2020 ).…”

Sensitive methods for detection of SARS-CoV-2 RNA

“…These formats include exo probe, nfo probe, fpg probe, digital ( Shen et al, 2011 ), nesting, microfluidics ( Lutz et al, 2010 ), solid phase, template generation, electrochemistry, colorimetric or SNP detection, and so on ( Daher, Stewart, Boissinot, & Bergeron, 2016 ; Li et al, 2020 ). Among all these, it seems that using exo probe for fluorescence detection ( Behrmann et al, 2020 ; Tu et al, 2020 ) and using nfo probe coupled with lateral flow strip detection ( Zheng et al, 2021 ) are the most popular and hence we will mainly focus on these two formats for the detection of SARS-CoV-2 viral RNA by RT-RPA. Herein, either approach has its own advantages.…”

“…Until now, there have been multiple reports about the detection of SARS-CoV-2 viral RNA using RT-RPA approach following our initial report in March 2020 and May 2020 ( Abd El Wahed et al, 2021 ; Arizti-Sanz et al, 2020 ; Behrmann et al, 2020 ; Qian et al, 2020 ; Tu et al, 2020 ; Wang, Cai, et al, 2020 ; Xia and Chen, 2020a , Xia and Chen, 2020b ; Xiong et al, 2020 ; Xue et al, 2020 ; Zheng et al, 2021 ). We introduced an WEPEAR ( w hole-course e ncapsulated p rocedure for e xponential a mplification from R NA) that seamlessly combines the reverse transcription (optimally at 37 °C, in the absence of Mg(OAc) 2 catalyst) and the RPA reaction (optimally at 40 °C, in the presence of Mg(OAc) 2 catalyst) at their own optimal conditions ( Fig.…”