Suitcase Lab for Rapid Detection of SARS-CoV-2 Based on Recombinase Polymerase Amplification Assay

Abstract

In March 2020, the SARS-CoV-2 virus outbreak was declared as a world pandemic by the World Health Organization (WHO). The only measures for controlling the outbreak are testing and isolation of infected cases. Molecular real-time polymerase chain reaction (PCR) assays are very sensitive but require highly equipped laboratories and well-trained personnel. In this study, a rapid point-of-need detection method was developed to detect the RNA-dependent RNA polymerase (RdRP), envelope protein (E), and nucleocapsid protein (N) genes of SARS-CoV-2 based on the reverse transcription recombinase polymerase amplification (RT-RPA) assay. RdRP, E, and N RT-RPA assays required approximately 15 min to amplify 2, 15, and 15 RNA molecules of molecular standard/reaction, respectively. RdRP and E RT-RPA assays detected SARS-CoV-1 and 2 genomic RNA, whereas the N RT-RPA assay identified only SARS-CoV-2 RNA. All established assays did not cross-react with nucleic acids of other respiratory pathogens. The RT-RPA assay’s clinical sensitivity and specificity in comparison to real-time RT-PCR (n = 36) were 94 and 100% for RdRP; 65 and 77% for E; and 83 and 94% for the N RT-RPA assay. The assays were deployed to the field, where the RdRP RT-RPA assays confirmed to produce the most accurate results in three different laboratories in Africa (n = 89). The RPA assays were run in a mobile suitcase laboratory to facilitate the deployment at point of need. The assays can contribute to speed up the control measures as well as assist in the detection of COVID-19 cases in low-resource settings.

Reverse Transcription Recombinase Polymerase Amplification Assay for Rapid Detection of Avian Influenza Virus H9N2 HA Gene

“…Many samples with very high CT values around 38 in real-time RT-PCR were identified as positive by RT-RPA in less than 7 min, which is an indication of the high sensitivity of the developed assay. A correlation between the CT values of the real-time RT-PCR and TT values of the RT-RPA was not observed due to the explosive nature of the RPA reaction, which outruns the repetitive, regular amplification cycles of the PCR reaction [ 30 ].…”

“…In conclusion, the developed H9 RT-RPA is rapid, simple, and sensitive for the on-site detection of H9N2 aIAVs. We have previously designed a “diagnostics-in-a-suitcase” for several pathogens [ 20 , 30 , 35 ], which can be applied for rapid detection of the virus under field settings, in order to start immediate control measures, nevertheless, according to national Egyptian law, all positive samples must be confirmed by governmental institutions.…”

Current diagnostic approaches to detect two important betacoronaviruses: Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

“…These findings implied that compared to other molecular targets, the N gene seems to be the target that is easier to be detected using the RT-RPA method [120] , [123] . When compared to qRT-PCR, RT-RPA seems to have lower sensitivity in which its sensitivity could vary from 65 to 94% while its specificity could range from 77 to 100% [124] . Therefore, it is said that RT-RPA could still be less sensitive and specific than qRT-PCR in confirming SARS-CoV-2 infection [124] .…”

“…When compared to qRT-PCR, RT-RPA seems to have lower sensitivity in which its sensitivity could vary from 65 to 94% while its specificity could range from 77 to 100% [124] . Therefore, it is said that RT-RPA could still be less sensitive and specific than qRT-PCR in confirming SARS-CoV-2 infection [124] .…”

Sensitive methods for detection of SARS-CoV-2 RNA

“…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.…”