Isothermal recombinase polymerase amplification-lateral flow detection of SARS-CoV-2, the etiological agent of COVID-19

Abstract

The rapid detection of novel pathogens including SARS-CoV-2 necessitates the development of easy-to-use diagnostic tests that can be readily adapted and utilized in both clinical laboratories and field settings. Delay in diagnosis has facilitated the rapid spread of this novel virus throughout the world resulting in global mortality that will surpass 2.5 million people. 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 cDNA 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 nasopharyngeal 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.

Keywords: Coronavirus; Diagnostics; Lateral flow; Point-of-care; RPA; SARS-CoV-2.

Fig. 1

Gene position and isothermal reaction results. A) Nucleotide schematic for N gene of SARS-CoV-2 with primer pair indicating relative positions of CDC N1-3. Selected primer pair for RPA-LF (N1 Fw and N3 Rv) are highlighted in red boxes. Green square depicts the position of RPA-LF probe. B) RPA-basic assessment of primer combinations at 40 °C.

Fig. 2

Specificity of SARS-CoV-2 RPA-LF. Selected primer pair for RPA-LF (N1 Fw and N3 Rv) and N1 RPA-LF probe were assessed at 42 °C for 40 min. No cross-reactivity was observed using 20,000 genome copies/reactionof SARS or MERS.

Fig. 3

Limit of detection using SARS-CoV-2 plasmid containing the nucleocapsid gene (copies/μL). Ten-fold serial dilutions of N gene plasmid compared to SARS and MERS plasmids that were used to test cross-reactivity of the RPA-LF and human RNase P plasmid (Hum RP) as the no virus control.

Fig. 4

Sensitivity of RPA-LF according to the incubation time at 42 °C. The assay has a limit of detection equivalent to 0.25 copies/μL of SARS-coV-2 since it was able to consistently amplify this cDNA concentration (5/6 total repetitions). Concentrations of 0.125 copies/μL yielded positive reactions in 2/3 repetitions.

Fig. 5

Genome equivalent limit of detection for SARS-CoV-2 RPA-LF. Concentration of genome equivalent was determined by standard curve of SARS-CoV-2 plasmid real-time PCR using the CDC N1 primer set. N1Fw and N3Rv were only able to detect 7.2 × 10³ genome equivalents per reaction by real-time PCR. Values are for 2.5 μL of input cDNA. (UD, undetermined; NTC, no template control; +, positive control; -, no virus control/Human Rnase P).

Fig. 6

Clinical efficacy of SARS-CoV-2 RPA-LF. Representative lateral flow strips of COVID-19 positive samples by RPA-LF, 1-11 and COVID-19 negative, 12-22. Numerical values represent corresponding Ct values for each sample; cut-off for positive samples is 37.5. (N.D., not detected; *, values greater than 37.5 are N.D.).