Reverse Transcription Recombinase-Aided Amplification Assay With Lateral Flow Dipstick Assay for Rapid Detection of 2019 Novel Coronavirus

Abstract

Background: The emerging Coronavirus Disease-2019 (COVID-19) has challenged the public health globally. With the increasing requirement of detection for SARS-CoV-2 outside of the laboratory setting, a rapid and precise Point of Care Test (POCT) is urgently needed.

Methods: Targeting the nucleocapsid (N) gene of SARS-CoV-2, specific primers, and probes for reverse transcription recombinase-aided amplification coupled with lateral flow dipstick (RT-RAA/LFD) platform were designed. For specificity evaluation, it was tested with human coronaviruses, human influenza A virus, influenza B viruses, respiratory syncytial virus, and hepatitis B virus, respectively. For sensitivity assay, it was estimated by templates of recombinant plasmid and pseudovirus of SARS-CoV-2 RNA. For clinical assessment, 100 clinical samples (13 positive and 87 negatives for SARS-CoV-2) were tested via quantitative reverse transcription PCR (RT-qPCR) and RT-RAA/LFD, respectively.

Results: The limit of detection was 1 copies/μl in RT-RAA/LFD assay, which could be conducted within 30 min at 39°C, without any cross-reaction with other human coronaviruses and clinical respiratory pathogens. Compared with RT-qPCR, the established POCT assay offered 100% specificity and 100% sensitivity in the detection of clinical samples.

Conclusion: This work provides a convenient POCT tool for rapid screening, diagnosis, and monitoring of suspected patients in SARS-CoV-2 endemic areas.

Keywords: Coronavirus Disease-2019; Severe Acute Respiratory Syndrome Coronavirus 2; lateral flow dipstick; point of care test; reverse-transcription recombinase-aided amplification.

Figure 1

Primers and probe design for RT-RAA/LFD. (A) Partial structure of the SARS-CoV-2 gene and locations of primers and probe. H indicates the tetrahydrofuran spacer (THF). Biotin indicates the Biotin label. PHO indicates phosphorylation blocking. FITC indicates the FITC fluorescein label. (B) Alignments of designed primers and probes and six coronaviruses. Dots indicate consensus, bars indicate absence.

Figure 2

Analytical specificity result of RT-RAA/LFD assay. RT-RAA/LFD specificity result. Blue for the control line while red for the test line. From left to right, the test samples are NEG for ribozyme-free water, 1 for H1N1, 2 for H3N2, 3 for influenza B viruses (Yamagata lineage), 4 for influenza B viruses (Victoria lineage), 5 for respiratory syncytial virus type A, 6 for respiratory syncytial virus type B, 7 for hepatitis B virus, 8 for HCoV-OC43, 9 for HCoV-HKU1, 10 for HCoV-NL63, 11 for HCoV-229E, 12 for SARS-CoV, POS for nucleic acid from SARS-CoV-2.

Figure 3

Analytical sensitivity result of RT-RAA/LFD for plasmid and SARS-CoV-2 RNA transcribed in vitro reference material (pseudovirus). Blue for the control line while red for the test line. (A) The sensitivity results of RT-RAA/LFD for plasmid. 10⁶ to 10⁰ indicates tests with 1 × 10⁶ to 1 copies/µl recombinant plasmid as a template. NEG indicates a test with ribozyme-free water as a template. (B) The sensitivity results of RT-RAA/LFD for SARS-CoV-2 RNA transcribed in vitro reference material. 10⁴ to 10⁰ indicates tests with 1 × 10⁴ to 1 copies/µl reference material as a template. NEG indicates a test with ribozyme-free water as a template.