Rapid detection of human coronavirus NL63 by isothermal reverse transcription recombinase polymerase amplification

Aline Dorendorf; Iris Bachmann; Martin Spiegel; Ahmed Abd El Wahed; Gregory Dame; Frank Hufert

doi:10.1016/j.jcvp.2022.100115

Rapid detection of human coronavirus NL63 by isothermal reverse transcription recombinase polymerase amplification

J Clin Virol Plus. 2022 Nov;2(4):100115. doi: 10.1016/j.jcvp.2022.100115. Epub 2022 Oct 8.

Authors

Aline Dorendorf¹, Iris Bachmann¹, Martin Spiegel^{1

2}, Ahmed Abd El Wahed³, Gregory Dame¹, Frank Hufert^{1

4

5}

Affiliations

¹ Institute of Microbiology and Virology, Brandenburg Medical School Theodor Fontane, Neuruppin, Germany.
² Infection Biology Unit, German Primate Center-Leibniz Institute for Primate Research, Göttingen, Germany.
³ Institute of Animal Hygiene and Veterinary Public Health, University of Leipzig, Germany.
⁴ Brandenburg University of Technology Cottbus, Senftenberg, Germany.
⁵ Faculty of Health Sciences, Joint Faculty of Brandenburg University of Technology Cottbus - Senftenberg, Brandenburg Medical School Theodor Fontane, Neuruppin and University of Potsdam, Potsdam, Germany.

Abstract

Background: Human coronaviruses are one of the leading causes for respiratory tract infections and for frequent primary care consultation. The human coronavirus NL63 (HCoV..µNL63) is one representative of the seasonal coronaviruses and capable of infecting the upper and lower respiratory tract and causative agent for croup in children.

Objectives: For fast detection of HCoV-NL63, we developed an isothermal reverse transcription recombinase polymerase amplification (RT-RPA) assay.

Study design: The analytical sensitivities of the RT-RPA assay were identified for in vitro transcribed ribonucleic acid (RNA) and for genomic viral RNA from cell culture supernatant. Moreover, specificity was tested with nucleic acids from other human coronaviruses and a variety of clinically relevant respiratory viruses. Finally, a clinical nasopharyngeal swab sample with spiked genomic viral HCoV-NL63 RNA was analyzed.

Results: Our HCoV-NL63 RT-RPA assay is highly specific and has an analytical sensitivity of 13 RNA molecules/reaction for in vitro transcribed RNA. For genomic viral RNA from cell culture supernatant spiked into a clinical nasopharyngeal swab sample the assay...s analytical sensitivity is 170 RNA molecules/reaction. The assay shows amplification of the lowest detectable target copy number after 8 minutes and 7 minutes, respectively.

Conclusions: We were able to design a sensitive and specific RT-RPA assay for the detection of HCoV-NL63. Additionally, the assay is characterized by short duration, isothermal amplification, and simple instrumentation.

Keywords: HCoV-NL63, human coronavirus NL63; LAMP, loop-mediated isothermal amplification; N-gene, nucleocapsid gene; RT-RPA, reverse transcription recombinase polymerase amplification; exo-IQ, internally quenched exo probe; human coronavirus NL63; recombinase polymerase amplification.