Establishment and application of a TaqMan-based multiplex real-time PCR for simultaneous detection of three porcine diarrhea viruses
IntroductionPorcine viral diarrhea is a common clinical disease, which results in high mortality and economic losses in the pig industry. Porcine epidemic diarrhea virus (PEDV), porcine rotavirus (PoRV), and porcine deltacoronavirus (PDCoV) are important diarrhea viruses in pig herds. The similariti...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2024-04-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2024.1380849/full |
| _version_ | 1797205901012107264 |
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| author | Jing Ren Jing Ren Congcong Zu Yang Li Meng Li Meng Li Jinyuan Gu Jinyuan Gu Fengling Chen Fengling Chen Xiaowen Li Xiaowen Li |
| author_facet | Jing Ren Jing Ren Congcong Zu Yang Li Meng Li Meng Li Jinyuan Gu Jinyuan Gu Fengling Chen Fengling Chen Xiaowen Li Xiaowen Li |
| author_sort | Jing Ren |
| collection | DOAJ |
| description | IntroductionPorcine viral diarrhea is a common clinical disease, which results in high mortality and economic losses in the pig industry. Porcine epidemic diarrhea virus (PEDV), porcine rotavirus (PoRV), and porcine deltacoronavirus (PDCoV) are important diarrhea viruses in pig herds. The similarities of their clinical symptoms and pathological changes make it difficult to distinguish these three viruses clinically. Therefore, there is a need for a highly sensitive and specific method to simultaneously detect and differentiate these viruses.MethodsA multiplex real-time PCR assay using TaqMan probes was developed to simultaneously detect PEDV, PoRV, and PDCoV. To assess the efficacy of the established assay, 30 clinical samples with diarrhea symptoms were used to compare the results obtained from the multiplex real-time PCR assay with those obtained from commercial singleplex real-time PCR kit. Importantly, a total of 4,800 diarrhea samples were tested and analyzed to validate the utility of the assay.ResultsThis multiplex real-time PCR assay showed high sensitivity, specificity, and excellent repeatability with a detection limit of 1 × 102 copies/μL. Comparing the results of the commercial singleplex real-time PCR kit and the multiplex real-time PCR method for detecting PEDV, PoRV, and PDCoV, there was complete agreement between the two approaches. Clinical data revealed single infection rates of 6.56% for PEDV, 21.69% for PoRV, and 6.65% for PDCoV. The co-infection rates were 11.83% for PEDV + PoRV, 0.29% for PEDV + PDCoV, 5.71% for PoRV + PDCoV, and 1.29% for PEDV + PDCoV + PoRV, respectively.DiscussionThe multiplex real-time PCR method established in this study is a valuable diagnostic tool for simultaneously differentiating PEDV, PoRV, and PDCoV. This method is expected to significantly contribute to prevent and control the spread of infectious diseases, as well as aid in conducting epidemiological investigations. |
| first_indexed | 2024-04-24T08:58:29Z |
| format | Article |
| id | doaj.art-4e18e2d7f0f5411595497f54e97b3e13 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-04-24T08:58:29Z |
| publishDate | 2024-04-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-4e18e2d7f0f5411595497f54e97b3e132024-04-16T05:00:33ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2024-04-011510.3389/fmicb.2024.13808491380849Establishment and application of a TaqMan-based multiplex real-time PCR for simultaneous detection of three porcine diarrhea virusesJing Ren0Jing Ren1Congcong Zu2Yang Li3Meng Li4Meng Li5Jinyuan Gu6Jinyuan Gu7Fengling Chen8Fengling Chen9Xiaowen Li10Xiaowen Li11Shandong Engineering Research Center of Swine Health Data and Intelligent Monitoring, Dezhou University, Dezhou, ChinaShandong Key Laboratory of Biophysics, Institute of Biophysics, Dezhou University, Dezhou, ChinaShandong New Hope Liuhe Agriculture and Animal Husbandry Technology Co., Ltd. (NHLH Academy of Swine Research), Dezhou, ChinaShandong New Hope Liuhe Agriculture and Animal Husbandry Technology Co., Ltd. (NHLH Academy of Swine Research), Dezhou, ChinaShandong Engineering Research Center of Swine Health Data and Intelligent Monitoring, Dezhou University, Dezhou, ChinaShandong Key Laboratory of Biophysics, Institute of Biophysics, Dezhou University, Dezhou, ChinaShandong Engineering Research Center of Swine Health Data and Intelligent Monitoring, Dezhou University, Dezhou, ChinaShandong Key Laboratory of Biophysics, Institute of Biophysics, Dezhou University, Dezhou, ChinaShandong Engineering Research Center of Swine Health Data and Intelligent Monitoring, Dezhou University, Dezhou, ChinaShandong Key Laboratory of Biophysics, Institute of Biophysics, Dezhou University, Dezhou, ChinaShandong Engineering Research Center of Swine Health Data and Intelligent Monitoring, Dezhou University, Dezhou, ChinaShandong New Hope Liuhe Agriculture and Animal Husbandry Technology Co., Ltd. (NHLH Academy of Swine Research), Dezhou, ChinaIntroductionPorcine viral diarrhea is a common clinical disease, which results in high mortality and economic losses in the pig industry. Porcine epidemic diarrhea virus (PEDV), porcine rotavirus (PoRV), and porcine deltacoronavirus (PDCoV) are important diarrhea viruses in pig herds. The similarities of their clinical symptoms and pathological changes make it difficult to distinguish these three viruses clinically. Therefore, there is a need for a highly sensitive and specific method to simultaneously detect and differentiate these viruses.MethodsA multiplex real-time PCR assay using TaqMan probes was developed to simultaneously detect PEDV, PoRV, and PDCoV. To assess the efficacy of the established assay, 30 clinical samples with diarrhea symptoms were used to compare the results obtained from the multiplex real-time PCR assay with those obtained from commercial singleplex real-time PCR kit. Importantly, a total of 4,800 diarrhea samples were tested and analyzed to validate the utility of the assay.ResultsThis multiplex real-time PCR assay showed high sensitivity, specificity, and excellent repeatability with a detection limit of 1 × 102 copies/μL. Comparing the results of the commercial singleplex real-time PCR kit and the multiplex real-time PCR method for detecting PEDV, PoRV, and PDCoV, there was complete agreement between the two approaches. Clinical data revealed single infection rates of 6.56% for PEDV, 21.69% for PoRV, and 6.65% for PDCoV. The co-infection rates were 11.83% for PEDV + PoRV, 0.29% for PEDV + PDCoV, 5.71% for PoRV + PDCoV, and 1.29% for PEDV + PDCoV + PoRV, respectively.DiscussionThe multiplex real-time PCR method established in this study is a valuable diagnostic tool for simultaneously differentiating PEDV, PoRV, and PDCoV. This method is expected to significantly contribute to prevent and control the spread of infectious diseases, as well as aid in conducting epidemiological investigations.https://www.frontiersin.org/articles/10.3389/fmicb.2024.1380849/fullmultiplex real-time PCRPEDVPoRVPDCoVporcine viral diarrhea |
| spellingShingle | Jing Ren Jing Ren Congcong Zu Yang Li Meng Li Meng Li Jinyuan Gu Jinyuan Gu Fengling Chen Fengling Chen Xiaowen Li Xiaowen Li Establishment and application of a TaqMan-based multiplex real-time PCR for simultaneous detection of three porcine diarrhea viruses Frontiers in Microbiology multiplex real-time PCR PEDV PoRV PDCoV porcine viral diarrhea |
| title | Establishment and application of a TaqMan-based multiplex real-time PCR for simultaneous detection of three porcine diarrhea viruses |
| title_full | Establishment and application of a TaqMan-based multiplex real-time PCR for simultaneous detection of three porcine diarrhea viruses |
| title_fullStr | Establishment and application of a TaqMan-based multiplex real-time PCR for simultaneous detection of three porcine diarrhea viruses |
| title_full_unstemmed | Establishment and application of a TaqMan-based multiplex real-time PCR for simultaneous detection of three porcine diarrhea viruses |
| title_short | Establishment and application of a TaqMan-based multiplex real-time PCR for simultaneous detection of three porcine diarrhea viruses |
| title_sort | establishment and application of a taqman based multiplex real time pcr for simultaneous detection of three porcine diarrhea viruses |
| topic | multiplex real-time PCR PEDV PoRV PDCoV porcine viral diarrhea |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2024.1380849/full |
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