Development of multiplex real-time PCR for simultaneous detection of SARS-CoV-2, CCoV, and FIPV

Abstract

Introduction: Coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), canine coronavirus (CCoV), and feline infectious peritonitis virus (FIPV), have the potential for interspecies transmission. These viruses can be present in complex environments where humans, dogs, and cats coexist, posing a significant threat to both human and animal safety.

Methods and results: In this study, we developed a novel multiplex TaqMan-probe-based real-time PCR assay for the simultaneous detection and differentiation of SARS-CoV-2, CCoV, and FIPV. Specific primers and TaqMan fluorescent probes were designed based on the N region of SARS-CoV-2 and FIPV, as well as the S region of CCoV, which demonstrated a remarkable sensitivity and specificity toward the targeted viruses, as few as 21.83, 17.25 and 9.25 copies/μL for SARS-CoV-2, CCoV and FIPV, respectively. The standard curve constructed by the optimized method in our present study showed a high amplification efficiency within or near the optimal range of 91% to 116% and R(2) values were at least 0.95 for the abovementioned coronaviruses. A total of 91 samples, including six plasmid mixed mock samples, four virus fluid mixing simulated samples, and 81 clinical samples, were analyzed using this method. Results demonstrated strong agreement with conventional approaches.

Discussion: By enabling the simultaneous detection of three viruses, this method enhances testing efficiency while decreasing costs. Importantly, it provides a valuable tool for the prevalence and geographical distribution of suspected and co-infected animals, ultimately contributing to the advancement of both animal and public health.

Keywords: FIPV; SARS-CoV-2; canine coronavirus; detection; real-time PCR.

Figure 1

(A) PCR identification of SA-N, CC-S, FI-N M: DL200 0/100 0 DNA Marker; 1: SA-N, CC-S, FI-N; 2: Negative control. (B) Digestion Identification of SA-N, CC-S, FI-N M: DL200 0/100 0 DNA Marker; 1: SA-N, CC-S, FI-N; 2: empty vector; 3: Negative control.

Figure 2

Preparation of plasmid standards. (A–C) Amplification curves (X-axis: Cycle, Y-axis: Fluorescence) of SARS-CoV-2, CCoV, and FIPV for each plasmid standard of concentrations with 2.183 × 10⁸copies/μL to 2.183 × 10⁴, 1.725 × 10¹⁰ to 1.725 × 10⁴ copies/μL, and 0.925 × 10¹⁰ to 0.925 × 10⁴copies/μL; (D–F) Standard curves of plasmid standards of SARS-CoV-2, CCoV, and FIPV. All standard curves were conducted with Microsoft Excel 2016.

Figure 3

(A–C) Amplification curves (X-axis: Cycle, Y-axis: Fluorescence) of SARS-CoV-2, CCoV, and FIPV detected by multiplex real-time PCR with different probe and primer concentrations. The three red lines are the amplification curves of fluorescence of the most suitable reaction tube.

Figure 4

(A–C) Amplification curves (X-axis: Cycle, Y-axis: Fluorescence) of SARS-CoV-2, CCoV, and FIPV detected by multiplex real-time PCR for each plasmid standard of concentrations with 2.183 × 10¹⁰ copies/μL to 2.183 × 10⁴, 1.725 × 10¹⁰ to 1.725 × 10⁴ copies/μL, and 0.925 × 10¹⁰ to 0.925 × 10⁴copies/μL; (D–F) Standard curves of plasmid standards of SARS-CoV-2, CCoV, and FIPV. All standard curves were conducted with Microsoft Excel 2016.

Figure 5

Sensitivity and specificity. (A–C) Amplification curves (X-axis: Cycle, Y-axis: Fluorescence) of SARS-CoV-2, CCoV, and FIPV detected by multiplex real-time PCR for each plasmid standard of concentrations with 2.183 × 10⁵copies/μL to 2.183 × 10¹, 1.725 × 10⁵ to 1.725 × 10¹ copies/μL, and 0.925 × 10⁵ to 0.925 × 10¹copies/μL; (D) Three amplification curves represent samples positive for SARS-CoV-2 (purple), CCoV (light blue), and FIPV (orange) detected by our multiplex real-time PCR assay; negative samples include FPV, FHV, FCV, CDV, CPV, ICHV, CAV-2, CPIV, and negative control.

Figure 6

Co-infection simulation experiments with two pathogens (SARS-CoV-2: purple, CCoV: light blue, FIPV: rosy). (A–C) Amplification curves (X-axis: Cycle, Y-axis: Fluorescence) of SARS-CoV-2 + CCoV, SARS-CoV-2 + FIPV, and CCoV + FIPV at concentrations of 1 × 10⁵copies/μL; (D–F) Amplification curves (X-axis: Cycle, Y-axis: Fluorescence) for viral mixtures of SARS-CoV-2 + CCoV, SARS-CoV-2 + FIPV, and CCoV + FIPV.

Figure 7

Co-infection simulation experiments with three pathogens (SARS-CoV-2: purple, CCoV: light blue, FIPV: orange or rosy). (A) The concentration of plasmid standard of SARS-CoV-2 was 10⁷ copies/μL and the others were 10² copies/μL; (B) The concentration of plasmid standard of CCoV was 10⁷ copies/μL and the others were 10² copies/μL; (C) The concentration of plasmid standard of FIPV was 10⁷ copies/μL and the others were 10² copies/μL; (D) amplification curves (X-axis: Cycle, Y-axis: Fluorescence) for viral mixtures of SARS-CoV-2 + CCoV + FIPV.