Development of a one-step multiplex RT-qPCR method for rapid detection of bovine diarrhea viruses
IntroductionViral calf diarrhea poses a significant challenge to the cattle industry worldwide due to its high morbidity and mortality rates, leading to substantial economic losses. The clinical symptoms associated with various diarrhea pathogens often overlap, complicating accurate diagnosis; thus,...
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Frontiers Media S.A.
2025-01-01
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| Series: | Frontiers in Cellular and Infection Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fcimb.2024.1540710/full |
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| author | Dequan Yang Li Ma Zhongping Yang Xianchao Yang Jian Wang Houbin Ju Chunguang Lu Yonggang Weng Heping Zhao Haixiao Shen Xin Li Feifei Ge Xiaoxu Wang Xiujuan Wu Meng Xiang Guidan Feng Congsheng Tang Shixin Huang Hongjin Zhao |
| author_facet | Dequan Yang Li Ma Zhongping Yang Xianchao Yang Jian Wang Houbin Ju Chunguang Lu Yonggang Weng Heping Zhao Haixiao Shen Xin Li Feifei Ge Xiaoxu Wang Xiujuan Wu Meng Xiang Guidan Feng Congsheng Tang Shixin Huang Hongjin Zhao |
| author_sort | Dequan Yang |
| collection | DOAJ |
| description | IntroductionViral calf diarrhea poses a significant challenge to the cattle industry worldwide due to its high morbidity and mortality rates, leading to substantial economic losses. The clinical symptoms associated with various diarrhea pathogens often overlap, complicating accurate diagnosis; thus, there is an urgent need for rapid and precise diagnostic methods to improve prevention and treatment efforts. In this study, we developed a one-step multiplex reverse-transcription quantitative real-time polymerase chain reaction (mRT-qPCR) that enables the simultaneous detection of three key viral pathogens responsible for calf diarrhea: bovine kobuvirus (BKoV), bovine astrovirus (BoAstV), and bovine torovirus (BToV). However, development of accurate and rapid methods to distinguish these three viruses is helpful for the early detection, disease surveillance, and control of viral calf diarrhea.MethodsSpecific primers and minor groove binder (MGB)-based probes were designed targeting the 3D region of BKoV, ORF1 region of BoAstV, and N region of BToV. The sensitivity, specificity, and reproducibility ability were evaluated for the mRT-qPCR. Further, 80 bovine fecal samples were subjected to the mRT-qPCR, and the results were verified using conventional reverse-transcription PCR (RT-PCR) or PCR methods and sequencing methods.ResultsThis novel method demonstrated high sensitivity and specificity,achieving a detection limit of 24 copies/mL for each pathogen. Furthermore, the assay exhibited excellent reproducibility, with coefficients of variation below 1.5%, a strong linear correlation (R2 > 0.996), and an amplification efficiency between 90% and 110%. Validation with 80 clinical samples from both diarrheic and non-diarrheic cattle across four farms in Shanghai showed a high degree of concordance with RT-PCR, with positive detection rates for BKoV, BoAstV, and BToV at 28.75%, 8.75%, and 3.75%, respectively, highlighting the predominance of BKoV and BoAstV. Notably, this study represents the first identification of BKoV, BoAstV, and BToV in the Shanghai region.DiscussionThe mRT-qPCR is a robust, rapid, and simple tool for identifying viral pathogens associated with calf diarrhea, facilitating the development of effective prevention and control measures that are vital for the future sustainability of the cattle industry. |
| format | Article |
| id | doaj-art-cb924f28f931450981bfbaf4444a75d6 |
| institution | Kabale University |
| issn | 2235-2988 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Cellular and Infection Microbiology |
| spelling | doaj-art-cb924f28f931450981bfbaf4444a75d62025-01-28T06:41:22ZengFrontiers Media S.A.Frontiers in Cellular and Infection Microbiology2235-29882025-01-011410.3389/fcimb.2024.15407101540710Development of a one-step multiplex RT-qPCR method for rapid detection of bovine diarrhea virusesDequan Yang0Li Ma1Zhongping Yang2Xianchao Yang3Jian Wang4Houbin Ju5Chunguang Lu6Yonggang Weng7Heping Zhao8Haixiao Shen9Xin Li10Feifei Ge11Xiaoxu Wang12Xiujuan Wu13Meng Xiang14Guidan Feng15Congsheng Tang16Shixin Huang17Hongjin Zhao18Veterinary Diagnostic Center, Shanghai Animal Disease Control Center, Shanghai, ChinaDepartment of Technological Research and Development, Hunan Guanmu Biotech Co., Ltd, Changsha, ChinaDepartment of Technological Research and Development, Hunan Guanmu Biotech Co., Ltd, Changsha, ChinaVeterinary Diagnostic Center, Shanghai Animal Disease Control Center, Shanghai, ChinaVeterinary Diagnostic Center, Shanghai Animal Disease Control Center, Shanghai, ChinaVeterinary Diagnostic Center, Shanghai Animal Disease Control Center, Shanghai, ChinaDepartment of Veterinary Laboratory, Jinshan District Animal Disease Control Center, Shanghai, ChinaDepartment of Veterinary Laboratory, Jinshan District Animal Disease Control Center, Shanghai, ChinaDepartment of Technological Research and Development, Hunan Guanmu Biotech Co., Ltd, Changsha, ChinaVeterinary Diagnostic Center, Shanghai Animal Disease Control Center, Shanghai, ChinaVeterinary Diagnostic Center, Shanghai Animal Disease Control Center, Shanghai, ChinaVeterinary Diagnostic Center, Shanghai Animal Disease Control Center, Shanghai, ChinaVeterinary Diagnostic Center, Shanghai Animal Disease Control Center, Shanghai, ChinaVeterinary Diagnostic Center, Shanghai Animal Disease Control Center, Shanghai, ChinaVeterinary Diagnostic Center, Shanghai Animal Disease Control Center, Shanghai, ChinaVeterinary Diagnostic Center, Shanghai Animal Disease Control Center, Shanghai, ChinaVeterinary Diagnostic Center, Shanghai Animal Disease Control Center, Shanghai, ChinaVeterinary Diagnostic Center, Shanghai Animal Disease Control Center, Shanghai, ChinaVeterinary Diagnostic Center, Shanghai Animal Disease Control Center, Shanghai, ChinaIntroductionViral calf diarrhea poses a significant challenge to the cattle industry worldwide due to its high morbidity and mortality rates, leading to substantial economic losses. The clinical symptoms associated with various diarrhea pathogens often overlap, complicating accurate diagnosis; thus, there is an urgent need for rapid and precise diagnostic methods to improve prevention and treatment efforts. In this study, we developed a one-step multiplex reverse-transcription quantitative real-time polymerase chain reaction (mRT-qPCR) that enables the simultaneous detection of three key viral pathogens responsible for calf diarrhea: bovine kobuvirus (BKoV), bovine astrovirus (BoAstV), and bovine torovirus (BToV). However, development of accurate and rapid methods to distinguish these three viruses is helpful for the early detection, disease surveillance, and control of viral calf diarrhea.MethodsSpecific primers and minor groove binder (MGB)-based probes were designed targeting the 3D region of BKoV, ORF1 region of BoAstV, and N region of BToV. The sensitivity, specificity, and reproducibility ability were evaluated for the mRT-qPCR. Further, 80 bovine fecal samples were subjected to the mRT-qPCR, and the results were verified using conventional reverse-transcription PCR (RT-PCR) or PCR methods and sequencing methods.ResultsThis novel method demonstrated high sensitivity and specificity,achieving a detection limit of 24 copies/mL for each pathogen. Furthermore, the assay exhibited excellent reproducibility, with coefficients of variation below 1.5%, a strong linear correlation (R2 > 0.996), and an amplification efficiency between 90% and 110%. Validation with 80 clinical samples from both diarrheic and non-diarrheic cattle across four farms in Shanghai showed a high degree of concordance with RT-PCR, with positive detection rates for BKoV, BoAstV, and BToV at 28.75%, 8.75%, and 3.75%, respectively, highlighting the predominance of BKoV and BoAstV. Notably, this study represents the first identification of BKoV, BoAstV, and BToV in the Shanghai region.DiscussionThe mRT-qPCR is a robust, rapid, and simple tool for identifying viral pathogens associated with calf diarrhea, facilitating the development of effective prevention and control measures that are vital for the future sustainability of the cattle industry.https://www.frontiersin.org/articles/10.3389/fcimb.2024.1540710/fullcalf diarrheamultiplex real-time quantitative PCR (mRT-qPCR)clinical detectionmethodepidemiological surveillance |
| spellingShingle | Dequan Yang Li Ma Zhongping Yang Xianchao Yang Jian Wang Houbin Ju Chunguang Lu Yonggang Weng Heping Zhao Haixiao Shen Xin Li Feifei Ge Xiaoxu Wang Xiujuan Wu Meng Xiang Guidan Feng Congsheng Tang Shixin Huang Hongjin Zhao Development of a one-step multiplex RT-qPCR method for rapid detection of bovine diarrhea viruses Frontiers in Cellular and Infection Microbiology calf diarrhea multiplex real-time quantitative PCR (mRT-qPCR) clinical detection method epidemiological surveillance |
| title | Development of a one-step multiplex RT-qPCR method for rapid detection of bovine diarrhea viruses |
| title_full | Development of a one-step multiplex RT-qPCR method for rapid detection of bovine diarrhea viruses |
| title_fullStr | Development of a one-step multiplex RT-qPCR method for rapid detection of bovine diarrhea viruses |
| title_full_unstemmed | Development of a one-step multiplex RT-qPCR method for rapid detection of bovine diarrhea viruses |
| title_short | Development of a one-step multiplex RT-qPCR method for rapid detection of bovine diarrhea viruses |
| title_sort | development of a one step multiplex rt qpcr method for rapid detection of bovine diarrhea viruses |
| topic | calf diarrhea multiplex real-time quantitative PCR (mRT-qPCR) clinical detection method epidemiological surveillance |
| url | https://www.frontiersin.org/articles/10.3389/fcimb.2024.1540710/full |
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