Isothermal Recombinase Polymerase Amplification-lateral Flow Detection of SARS-CoV-2, the Etiological Agent of COVID-19

Abstract

The rapid detection of novel pathogens necessitates the development of easy-to-use diagnostic tests that can be readily adapted and utilized in both clinical laboratories and field settings. In December of 2019, novel coronavirus, SARS-CoV-2 (2019-nCoV), was isolated from a cluster of pneumonia patients in the Chinese city of Wuhan. The virus rapidly spread throughout the world and the first fatal cases of COVID-19 in the United States occurred in late February. The lack of testing and delay in diagnosis has facilitated the spread of this novel virus. Development of point-of-care diagnostic assays that can be performed in rural or decentralized health care centers to expand testing capacity is needed. We developed a qualitative test based on recombinase-polymerase-amplification coupled with lateral flow reading (RPA-LF) for rapid detection of SARS-CoV-2. The RPA-LF detected SARS-CoV-2 with a limit of detection of 35.4 viral nucleocapsid (N) gene copies/µL. Additionally, the RPA-LF was able to detect 0.25-2.5 copies/µL of SARS-CoV-2 N gene containing plasmid. We evaluated 37 clinical samples using CDC’s N3, N1 and N2 RT-real-time PCR assays for SARS-CoV-2 as reference test. We found a 100% concordance between RPA-LF and RT-qPCR reference test as determined by 18/18 positive and 19/19 negative samples. All positive samples had Ct values between 19-37 by RT-qPCR. The RPA-LF primers and probe did not cross react with other relevant betacoronaviruses such as SARS and MERS. This is the first isothermal amplification test paired with lateral flow developed for qualitative detection of COVID-19 allowing rapid viral detection and with prospective applicability in resource limited and decentralized laboratories.

Recombinase Polymerase Amplification for Rapid Detection of Zoonotic Pathogens

“…The development of sensitive, rapid, specific and cost-effective detection methods has never been more important. RPA has been widely used for the detection of SARS-CoV-2 viral RNA in clinical samples [71,97,125]. Combine with the lateral flow assays, reverse transcriptase recombinase polymerase amplification can complete the detection of pathogen nucleic acids within 20min and the detection limit can be as low as 7.659 copies/μL RNA [69].…”

CoVrimer: A tool for aligning SARS-CoV-2 primer sequences and selection of conserved/degenerate primers

“…Relatively less costly detection methods are also available, including SYBR Green and conventional RT-PCR [8]. Other detection strategies include reverse transcription loop-mediated isothermal amplification (RT-LAMP) [9][10][11], reverse transcription recombinase-aided amplification (RT-RAA) [12,13], and recombinase polymerase amplification-lateral flow reading (RPA-LF) [14] assays as well as those based on sequencing [15], droplet digital PCR (ddPCR) [16], and RT-LAMP-CRISPR platform [17]. Despite the abundance of diagnostic methods, the quantitative nature of qRT-PCR may be necessary when especially the information on viral load is required [11].…”