Development of a multi-recombinase polymerase amplification assay for rapid identification of COVID-19, influenza A and B

Abstract

The coronavirus disease 2019 (COVID-19) pandemic caused extensive loss of life worldwide. Further, the COVID-19 and influenza mix-infection had caused great distress to the diagnosis of the disease. To control illness progression and limit viral spread within the population, a real-time reverse-transcription PCR (RT-PCR) assay for early diagnosis of COVID-19 was developed, but detection was time-consuming (4-6 h). To improve the diagnosis of COVID-19 and influenza, we herein developed a recombinase polymerase amplification (RPA) method for simple and rapid amplification of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19 and Influenza A (H1N1, H3N2) and B (influenza B). Genes encoding the matrix protein (M) for H1N1, and the hemagglutinin (HA) for H3N2, and the polymerase A (PA) for Influenza B, and the nucleocapsid protein (N), the RNA-dependent-RNA polymerase (RdRP) in the open reading frame 1ab (ORF1ab) region, and the envelope protein (E) for SARS-CoV-2 were selected, and specific primers were designed. We validated our method using SARS-CoV-2, H1N1, H3N2 and influenza B plasmid standards and RNA samples extracted from COVID-19 and Influenza A/B (RT-PCR-verified) positive patients. The method could detect SARS-CoV-2 plasmid standard DNA quantitatively between 10² and 10⁵ copies/ml with a log linearity of 0.99 in 22 min. And this method also be very effective in simultaneous detection of H1N1, H3N2 and influenza B. Clinical validation of 100 cases revealed a sensitivity of 100% for differentiating COVID-19 patients from healthy controls when the specificity was set at 90%. These results demonstrate that this nucleic acid testing method is advantageous compared with traditional PCR and other isothermal nucleic acid amplification methods in terms of time and portability. This method could potentially be used for detection of SARS-CoV-2, H1N1, H3N2 and influenza B, and adapted for point-of-care (POC) detection of a broad range of infectious pathogens in resource-limited settings.

Keywords: COVID-19; RPA; SARS-CoV-2; influenza; laboratory diagnosis.

Figure 1

The principle of RPA test for screening of COVID‐19 and influenza patients. (A) Nasopharyngeal swab samples that may contain virus were obtained from patients by medical staff. (B) The viral nucleic acid from the samples was obtained according to the instruction of viral RNA extraction. (C) Following RNA extraction, the RNA samples were screening via an RPA method in Octet PCR tube. (D) The amplification data were sent to a laptop data analysis. COVID‐19, coronavirus disease 2019; PCR, polymerase chain reaction; RPA, recombinase polymerase amplification

Figure 2

Sensitivity of SARS‐CoV‐2, H1N1, H3N2 and IVB by RPA assay. (A–C) Sensitivity of the RPA assay for ORF1ab, N, and E gene of SARS‐CoV‐2. (D) Sensitivity of the RPA assay for M gene of H1N1. (E) Sensitivity of the RPA assay for HA gene of H3N2. (F) Sensitivity of the RPA assay for PA gene of IVB. RPA, recombinase polymerase amplification; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2.

Figure 3

Clinical validation of ORF1ab, E, N, M, HA, PA genes for differentiating COVID‐19 and influenza patients from healthy individuals. (A) 70 RNA samples from COVID‐19 patients were amplified using the RPA method. (B and C) 55 RNA samples from influenza A patients (H1N1, n‐20; H3N2, n = 20) were amplified using the RPA method developed herein. (D) 15 RNA samples from influenza B patients were amplified via the RPA method. COVID‐19, coronavirus disease 2019; RPA, recombinase polymerase amplification.

Figure 4

Box‐plot and receiver operating characteristic (ROC) curve analyses. The Cq value of RNA samples from COVID‐19 patients was significantly lower than that from healthy controls (A, C, E). In addition, a ROC curve was plotted for clinical validation, and the RPA assay had a sensitivity of 100% at a specificity of 96.67%, 96.67%, and 100% for N, ORF1ab and E gene, respectively for identifying COVID‐19 patients from healthy controls, with an AUROC value of 0.99 (B, D, F). COVID‐19, coronavirus disease 2019; RPA, recombinase polymerase amplification.

Figure 5

Comparison of amplification results for qPCR and RPA. To compare the amplification effect for RPA and real‐time PCR, 30 samples of COVID‐19 patients were performed the experiment. The Cq value was shown the ORF1ab (A), N (C), and E (E) gene of SARS‐CoV‐2. The time value was shown the ORF1ab (B), N (D), and E (F) gene of SARS‐CoV‐2. These results showed that the detection time of RPA was much faster than that of real‐time PCR. COVID‐19, coronavirus disease 2019; RPA, recombinase polymerase amplification; RT‐PCR, reverse‐transcription polymerase chain reaction.