A Quadruplex RT-qPCR for the Detection of Porcine Sapelovirus, Porcine Kobuvirus, Porcine Teschovirus, and Porcine Enterovirus G
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, an...
Saved in:
| Main Authors: | , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2025-03-01
|
| Series: | Animals |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2076-2615/15/7/1008 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1849730967081582592 |
|---|---|
| author | Biao Li Kaichuang Shi Yuwen Shi Shuping Feng Yanwen Yin Wenjun Lu Feng Long Zuzhang Wei Yingyi Wei |
| author_facet | Biao Li Kaichuang Shi Yuwen Shi Shuping Feng Yanwen Yin Wenjun Lu Feng Long Zuzhang Wei Yingyi Wei |
| author_sort | Biao Li |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-3dbb45282cd347d0a42e0eb22fa10a61 |
| institution | DOAJ |
| issn | 2076-2615 |
| language | English |
| publishDate | 2025-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Animals |
| spelling | doaj-art-3dbb45282cd347d0a42e0eb22fa10a612025-08-20T03:08:43ZengMDPI AGAnimals2076-26152025-03-01157100810.3390/ani15071008A Quadruplex RT-qPCR for the Detection of Porcine Sapelovirus, Porcine Kobuvirus, Porcine Teschovirus, and Porcine Enterovirus GBiao Li0Kaichuang Shi1Yuwen Shi2Shuping Feng3Yanwen Yin4Wenjun Lu5Feng Long6Zuzhang Wei7Yingyi Wei8Guangxi Key Laboratory of Animal Breeding, Disease Control and Prevention, College of Animal Science and Technology, Guangxi University, Nanning 530005, ChinaGuangxi Key Laboratory of Animal Breeding, Disease Control and Prevention, College of Animal Science and Technology, Guangxi University, Nanning 530005, ChinaGuangxi Key Laboratory of Animal Breeding, Disease Control and Prevention, College of Animal Science and Technology, Guangxi University, Nanning 530005, ChinaGuangxi Center for Animal Disease Control and Prevention, Nanning 530001, ChinaGuangxi Center for Animal Disease Control and Prevention, Nanning 530001, ChinaGuangxi Center for Animal Disease Control and Prevention, Nanning 530001, ChinaGuangxi Center for Animal Disease Control and Prevention, Nanning 530001, ChinaGuangxi Key Laboratory of Animal Breeding, Disease Control and Prevention, College of Animal Science and Technology, Guangxi University, Nanning 530005, ChinaGuangxi Key Laboratory of Animal Breeding, Disease Control and Prevention, College of Animal Science and Technology, Guangxi University, Nanning 530005, ChinaPorcine 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.https://www.mdpi.com/2076-2615/15/7/1008porcine sapelovirus (PSV)porcine kobuvirus (PKV)porcine teschovirus (PTV)porcine enterovirus G (EV-G)multiplex RT-qPCRco-infection |
| spellingShingle | Biao Li Kaichuang Shi Yuwen Shi Shuping Feng Yanwen Yin Wenjun Lu Feng Long Zuzhang Wei Yingyi Wei A Quadruplex RT-qPCR for the Detection of Porcine Sapelovirus, Porcine Kobuvirus, Porcine Teschovirus, and Porcine Enterovirus G Animals porcine sapelovirus (PSV) porcine kobuvirus (PKV) porcine teschovirus (PTV) porcine enterovirus G (EV-G) multiplex RT-qPCR co-infection |
| title | A Quadruplex RT-qPCR for the Detection of Porcine Sapelovirus, Porcine Kobuvirus, Porcine Teschovirus, and Porcine Enterovirus G |
| title_full | A Quadruplex RT-qPCR for the Detection of Porcine Sapelovirus, Porcine Kobuvirus, Porcine Teschovirus, and Porcine Enterovirus G |
| title_fullStr | A Quadruplex RT-qPCR for the Detection of Porcine Sapelovirus, Porcine Kobuvirus, Porcine Teschovirus, and Porcine Enterovirus G |
| title_full_unstemmed | A Quadruplex RT-qPCR for the Detection of Porcine Sapelovirus, Porcine Kobuvirus, Porcine Teschovirus, and Porcine Enterovirus G |
| title_short | A Quadruplex RT-qPCR for the Detection of Porcine Sapelovirus, Porcine Kobuvirus, Porcine Teschovirus, and Porcine Enterovirus G |
| title_sort | quadruplex rt qpcr for the detection of porcine sapelovirus porcine kobuvirus porcine teschovirus and porcine enterovirus g |
| topic | porcine sapelovirus (PSV) porcine kobuvirus (PKV) porcine teschovirus (PTV) porcine enterovirus G (EV-G) multiplex RT-qPCR co-infection |
| url | https://www.mdpi.com/2076-2615/15/7/1008 |
| work_keys_str_mv | AT biaoli aquadruplexrtqpcrforthedetectionofporcinesapelovirusporcinekobuvirusporcineteschovirusandporcineenterovirusg AT kaichuangshi aquadruplexrtqpcrforthedetectionofporcinesapelovirusporcinekobuvirusporcineteschovirusandporcineenterovirusg AT yuwenshi aquadruplexrtqpcrforthedetectionofporcinesapelovirusporcinekobuvirusporcineteschovirusandporcineenterovirusg AT shupingfeng aquadruplexrtqpcrforthedetectionofporcinesapelovirusporcinekobuvirusporcineteschovirusandporcineenterovirusg AT yanwenyin aquadruplexrtqpcrforthedetectionofporcinesapelovirusporcinekobuvirusporcineteschovirusandporcineenterovirusg AT wenjunlu aquadruplexrtqpcrforthedetectionofporcinesapelovirusporcinekobuvirusporcineteschovirusandporcineenterovirusg AT fenglong aquadruplexrtqpcrforthedetectionofporcinesapelovirusporcinekobuvirusporcineteschovirusandporcineenterovirusg AT zuzhangwei aquadruplexrtqpcrforthedetectionofporcinesapelovirusporcinekobuvirusporcineteschovirusandporcineenterovirusg AT yingyiwei aquadruplexrtqpcrforthedetectionofporcinesapelovirusporcinekobuvirusporcineteschovirusandporcineenterovirusg AT biaoli quadruplexrtqpcrforthedetectionofporcinesapelovirusporcinekobuvirusporcineteschovirusandporcineenterovirusg AT kaichuangshi quadruplexrtqpcrforthedetectionofporcinesapelovirusporcinekobuvirusporcineteschovirusandporcineenterovirusg AT yuwenshi quadruplexrtqpcrforthedetectionofporcinesapelovirusporcinekobuvirusporcineteschovirusandporcineenterovirusg AT shupingfeng quadruplexrtqpcrforthedetectionofporcinesapelovirusporcinekobuvirusporcineteschovirusandporcineenterovirusg AT yanwenyin quadruplexrtqpcrforthedetectionofporcinesapelovirusporcinekobuvirusporcineteschovirusandporcineenterovirusg AT wenjunlu quadruplexrtqpcrforthedetectionofporcinesapelovirusporcinekobuvirusporcineteschovirusandporcineenterovirusg AT fenglong quadruplexrtqpcrforthedetectionofporcinesapelovirusporcinekobuvirusporcineteschovirusandporcineenterovirusg AT zuzhangwei quadruplexrtqpcrforthedetectionofporcinesapelovirusporcinekobuvirusporcineteschovirusandporcineenterovirusg AT yingyiwei quadruplexrtqpcrforthedetectionofporcinesapelovirusporcinekobuvirusporcineteschovirusandporcineenterovirusg |