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

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.

Fig. 1

Real-time RT-RPA sensitivity assay with synthetic HCoV-NL63 RNA. A) Fluorescence over time was measured in an RT-RPA running at 42 °C for 20 min. Each standard concentration containing synthetic HCoV-NL63 RNA (10⁷ RNA molecules/reaction represented by dark blue line; 10⁶, yellow; 10⁵, purple; 10⁴, black; 10³, light blue; 10², magenta; 10¹, orange) as well as PCR-grade water as no template-control (NTC; grey line) was assessed in 8 replicates (n=8). Normalized data are shown, and threshold is indicated as dotted black line. B) Calibration line for the detection of HCoV-NL63 RNA in a real-time RT-RPA assay. Shown is the linear correlation of threshold times over the decadic logarithm of the RNA molecules/reaction. C) Probit regression for synthetic HCOV-NL63 RNA in PCR-grade water revealing a 95 % detection probability (analytical sensitivity) of 13 RNA copies/reaction (dotted red line).

Fig. 2

Real-time RT-RPA sensitivity assay with genomic viral HCoV-NL63 RNA in human nasopharyngeal background. A) Fluorescence over time was measured in an RT-RPA running at 42 °C for 20 min. Each standard concentration containing viral HCoV-NL63 RNA from cell culture and human RNA from swab sample material (5 × 10⁶ RNA molecules/reaction represented by dark blue line; 5 × 10⁵, yellow; 5 × 10⁴, purple; 5 × 10³, black; 5 × 10², light blue; 5 × 10¹, magenta; 5 × 10⁰, orange) as well as PCR-grade water as no template-control (NTC; grey line) was assessed in 8 replicates (n=8) and 2 replicates (n=2) containing 5 × 10² synthetic RNA molecules/reaction as positive control (dotted light blue line). Normalized data are shown, and threshold is indicated as dotted black line. B) Calibration line for the detection of viral HCoV-NL63 RNA in a real-time RT-RPA assay. Shown is the linear correlation of threshold times over the decadic logarithm of the RNA target concentration. C) Probit regression for viral HCoV-NL63 RNA from cell culture and human RNA from swab sample material revealing a 95 % detection probability (analytical sensitivity) of 170 RNA molecules/reaction.