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

• Published in: Journal of clinical virology plus Vol. 2; no. 4; p. 100115
• Main Authors: Dorendorf, Aline, Bachmann, Iris, Spiegel, Martin, Abd El Wahed, Ahmed, Dame, Gregory, Hufert, Frank
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.11.2022; The Authors. Published by Elsevier Ltd; Elsevier
• Subjects: human coronavirus NL63; recombinase polymerase amplification; N-gene; recombinase polymerase amplification; human coronavirus NL63; HCoV-NL63; RT-RPA; LAMP; exo-IQ; RT-RPA, reverse transcription recombinase polymerase amplification; HCoV-NL63, human coronavirus NL63; LAMP, loop-mediated isothermal amplification; exo-IQ, internally quenched exo probe; N-gene, nucleocapsid gene
• ISSN: 2667-0380; 2667-0380
• DOI: 10.1016/j.jcvp.2022.100115

•Isothermal amplification assay for the detection of human coronavirus NL63 RNA in less than 15 minutes.•Specific molecular detection of NL63 coronavirus.•Detection of NL63 coronavirus in spiked human swab sample material. 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. For fast detection of HCoV-NL63, we developed an isothermal reverse transcription recombinase polymerase amplification (RT-RPA) assay. 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. 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. 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.