A Quadruplex RT-qPCR for the Detection of Porcine Sapelovirus, Porcine Kobuvirus, Porcine Teschovirus, and Porcine Enterovirus G

Abstract

Porcine sapelovirus (PSV), porcine kobuvirus (PKV), porcine teschovirus (PTV), and porcine enterovirus G (EV-G) are all important viruses in the swine industry. These viruses play important roles in the establishment of similar clinical signs of diseases in pigs, including diarrhea, encephalitis, and reproductive and respiratory disorders. The early accurate detection of these viruses is crucial for dealing with these diseases. In order for the differential detection of these four viruses, specific primers and TaqMan probes were designed for the conserved regions in the 5' untranslated region (UTR) of these four viruses, and one-step quadruplex reverse-transcription real-time quantitative PCR (RT-qPCR) for the detection of PSV, PKV, PTV, and EV-G was developed. The results showed that this assay had the advantages of high sensitivity, strong specificity, excellent repeatability, and simple operation. Probit regression analysis showed that the assay obtained low limits of detection (LODs) for PSV, PKV, PTV, and EV-G, with 146.02, 143.83, 141.92, and 139.79 copies/reaction, respectively. The assay showed a strong specificity of detecting only PSV, PKV, PTV, and EV-G, and had no cross-reactivity with other control viruses. The assay exhibited excellent repeatability of the intra-assay coefficient of variation (CV) of 0.28-1.58% and the inter-assay CV of 0.20-1.40%. Finally, the developed quadruplex RT-qPCR was used to detect 1823 fecal samples collected in Guangxi Province, China between January 2024 and December 2024. The results indicated that the positivity rates of PSV, PKV, PTV, and EV-G were 15.25% (278/1823), 21.72% (396/1823), 18.82% (343/1823), and 27.10% (494/1823), respectively, and there existed phenomena of mixed infections. Compared with the reference RT-qPCR/RT-PCR established for these four viruses, the coincidence rates for the detection results of PSV, PKV, PTV, and EV-G reached 99.51%, 99.40%, 99.51%, and 99.01%, respectively. In conclusions, the developed quadruplex RT-qPCR could simultaneously detect PSV, PKV, PTV, and EV-G, and provided an efficient and convenient detection method to monitor the epidemic status and variation of these viruses.

Keywords: co-infection; multiplex RT-qPCR; porcine enterovirus G (EV-G); porcine kobuvirus (PKV); porcine sapelovirus (PSV); porcine teschovirus (PTV).

Figure 1

The amplification curves of p-PSV (A), p-PKV (B), p-PTV (C), and p-EV-G (D), and the standard curves (E) of the developed quadruplex RT-qPCR. In subfigure (A–D) 1–7, the final concentration of the plasmid constructs ranged from 10⁷ to 10¹ copies/$\mathrm{\mu }\mathrm{L}$; 8: distilled water.

Figure 2

Specificity evaluation of the developed quadruplex RT-qPCR for PSV (A), PKV (B), PTV (C), and EV-G (D). 1: p-PSV; 2: p-PKV; 3: p-PTV; 4: p-EV-G; 5–8: positive clinical samples of PSV, PKV, PTV, and EV-G; 9: PSV; 10: PKV; 11: PTV; 12: EV-G; 13–22: TGEV, PCV2, PRRSV, FMDV, CSFV, PRV, SIV, PoRV, ASFV, PEDV, and PDCoV; 23: negative fecal sample; 24: distilled water.

Figure 3

Sensitivity evaluation of the developed quadruplex RT-qPCR. In subfigures (A–D) 1–8: the final reaction concentrations of the synthesized viral RNAs ranged from 10⁷ to 10⁰ copies/$\mathrm{\mu }\mathrm{L}$; 9: negative control.

Figure 4

Sensitivity evaluation of the developed quadruplex RT-qPCR using probit regression analysis. The PSV (A), PKV (B), PTV (C), and EV-G (D) had LODs of 146.02, 143.83, 141.92, and 139.79 copies/reaction, respectively.