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Updated: Aug 29, 2025
Author Spotlight: Advancements in Multiplex Detection of Respiratory Viruses
Published on: November 10, 2023
Development of a multi-recombinase polymerase amplification assay for rapid identification of COVID-19, influenza A
Li-Guo Liang1,2,3, Miao-Jin Zhu1,2, Rui He4
1State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
Insights
A new recombinase polymerase amplification (RPA) method rapidly detects Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and influenza viruses. This rapid nucleic acid test offers a portable solution for simultaneous diagnosis of COVID-19 and influenza, improving patient outcomes.
Area of Science:
- Molecular Biology
- Virology
- Diagnostic Assays
Background:
- The COVID-19 pandemic caused significant global mortality, complicated by co-infections with influenza.
- Existing diagnostic methods like RT-PCR for COVID-19 are time-consuming (4-6 hours), hindering rapid diagnosis and control.
- Accurate and swift differentiation between COVID-19 and influenza infections is crucial for effective patient management and limiting viral spread.
Purpose of the Study:
- To develop a simple, rapid, and accurate diagnostic method for simultaneous detection of SARS-CoV-2 and influenza A/B viruses.
- To overcome the time limitations associated with conventional RT-PCR assays.
- To create a potentially portable assay suitable for point-of-care applications, especially in resource-limited settings.
Main Methods:
- Developed a recombinase polymerase amplification (RPA) method for nucleic acid amplification.
- Designed specific primers targeting key genes of SARS-CoV-2 (N, RdRP, E genes) and influenza strains (M for H1N1, HA for H3N2, PA for Influenza B).
- Validated the RPA assay using plasmid standards and clinical RNA samples from confirmed COVID-19 and influenza patients.
Main Results:
- The RPA method quantitatively detected SARS-CoV-2 plasmid DNA (10^2–10^5 copies/ml) with high linearity (R^2=0.99) in just 22 minutes.
- Demonstrated effective simultaneous detection capabilities for influenza A (H1N1, H3N2) and influenza B.
- Clinical validation in 100 cases showed 100% sensitivity for differentiating COVID-19 patients from healthy controls at 90% specificity.
Conclusions:
- The developed RPA assay offers a significant improvement over traditional PCR and other isothermal methods in terms of speed and portability.
- This nucleic acid testing method is highly effective for the simultaneous detection of SARS-CoV-2 and common influenza strains.
- The assay holds potential for widespread application in point-of-care diagnostics for infectious diseases, particularly in resource-limited environments.
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.
