Establishment and application of a one-step multiplex real-time PCR assay for detection of A, B, and C subtypes of avian metapneumovirus
Avian metapneumovirus (aMPV) represents a significant threat to the poultry industry, exhibiting a high degree of genetic diversity. Of these, the aMPV types A (aMPV-A), B (aMPV-B) and C (aMPV-C) are frequently detected in Chinese waterfowl and live poultry markets. Therefore, the rapid and accurate...
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Elsevier
2025-01-01
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| Series: | Poultry Science |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S0032579124011866 |
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| author | Mingjun Su Jiongze Cheng Xiangwen Xu Yijia Liu Yulin Zhao Yutao Wang Xiaoxu Du Jiale Ying Junfang Yan Huihua Zheng Changyong Cheng Jing Sun |
| author_facet | Mingjun Su Jiongze Cheng Xiangwen Xu Yijia Liu Yulin Zhao Yutao Wang Xiaoxu Du Jiale Ying Junfang Yan Huihua Zheng Changyong Cheng Jing Sun |
| author_sort | Mingjun Su |
| collection | DOAJ |
| description | Avian metapneumovirus (aMPV) represents a significant threat to the poultry industry, exhibiting a high degree of genetic diversity. Of these, the aMPV types A (aMPV-A), B (aMPV-B) and C (aMPV-C) are frequently detected in Chinese waterfowl and live poultry markets. Therefore, the rapid and accurate identification of these subtypes is of paramount importance in order to halt the spread of the disease. In this study, we have developed a multiplex real-time PCR assay endowed with the capacity to simultaneously discriminate aMPV-A, aMPV-B, and aMPV-C. This method demonstrates remarkable specificity, selectively amplifying aMPV-A, aMPV-B, and aMPV-C without cross-reactivity with other common avian pathogens. Furthermore, this method exhibits high sensitivity, with a detection threshold of 8.5 × 102 copies/μL for aMPV-A, aMPV-B, and aMPV-C. Moreover, the assay demonstrates reproducibility, as evidenced by intra- and inter-assay variability, with a coefficient of variation between 0.21% and 1.91%. Additionally, the receiver operating characteristic (ROC) curve analysis demonstrated that the multiplex real-time PCR assay exhibited high specificity and sensitivity (100.0% and 100.0% for aMPV-A, 90.9% and 100.0% for aMPV-B, 100% and 96.8% for aMPV-C) when compared with the classical aMPV real-time RT-PCR. Analyses of field samples (n=105) using the multiplex real-time PCR assay indicated that 35.2% (37/105) of samples were positive for aMPV, of which 29.7% (11/37) for aMPV-A, 32.4% (12/37) for aMPV-B and 37.8% (14/37) for aMPV-C. These data demonstrated that the multiplex real-time PCR assay can be used for epidemiological investigations of tree subtypes of aMPV and that aMPV had been observed to exhibit a proclivity for multiple types of co-infection in the Zhejiang province of China. |
| format | Article |
| id | doaj-art-59a7717399094aacb7ca10312a86edb1 |
| institution | Kabale University |
| issn | 0032-5791 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Elsevier |
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| series | Poultry Science |
| spelling | doaj-art-59a7717399094aacb7ca10312a86edb12025-01-22T05:40:41ZengElsevierPoultry Science0032-57912025-01-011041104608Establishment and application of a one-step multiplex real-time PCR assay for detection of A, B, and C subtypes of avian metapneumovirusMingjun Su0Jiongze Cheng1Xiangwen Xu2Yijia Liu3Yulin Zhao4Yutao Wang5Xiaoxu Du6Jiale Ying7Junfang Yan8Huihua Zheng9Changyong Cheng10Jing Sun11Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, Zhejiang Provincial Engineering Research Center for Animal Health Diagnostics & Advanced Technology, Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management, China-Australia Joint Laboratory for Animal Health Big Data Analytics, College of Animal Science and Technology & College of Veterinary Medicine of Zhejiang A&F University, 666 Wusu Street, Lin'an District, Hangzhou, Zhejiang Province 311300, ChinaKey Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, Zhejiang Provincial Engineering Research Center for Animal Health Diagnostics & Advanced Technology, Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management, China-Australia Joint Laboratory for Animal Health Big Data Analytics, College of Animal Science and Technology & College of Veterinary Medicine of Zhejiang A&F University, 666 Wusu Street, Lin'an District, Hangzhou, Zhejiang Province 311300, ChinaKey Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, Zhejiang Provincial Engineering Research Center for Animal Health Diagnostics & Advanced Technology, Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management, China-Australia Joint Laboratory for Animal Health Big Data Analytics, College of Animal Science and Technology & College of Veterinary Medicine of Zhejiang A&F University, 666 Wusu Street, Lin'an District, Hangzhou, Zhejiang Province 311300, ChinaKey Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, Zhejiang Provincial Engineering Research Center for Animal Health Diagnostics & Advanced Technology, Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management, China-Australia Joint Laboratory for Animal Health Big Data Analytics, College of Animal Science and Technology & College of Veterinary Medicine of Zhejiang A&F University, 666 Wusu Street, Lin'an District, Hangzhou, Zhejiang Province 311300, ChinaKey Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, Zhejiang Provincial Engineering Research Center for Animal Health Diagnostics & Advanced Technology, Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management, China-Australia Joint Laboratory for Animal Health Big Data Analytics, College of Animal Science and Technology & College of Veterinary Medicine of Zhejiang A&F University, 666 Wusu Street, Lin'an District, Hangzhou, Zhejiang Province 311300, ChinaKey Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, Zhejiang Provincial Engineering Research Center for Animal Health Diagnostics & Advanced Technology, Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management, China-Australia Joint Laboratory for Animal Health Big Data Analytics, College of Animal Science and Technology & College of Veterinary Medicine of Zhejiang A&F University, 666 Wusu Street, Lin'an District, Hangzhou, Zhejiang Province 311300, ChinaKey Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, Zhejiang Provincial Engineering Research Center for Animal Health Diagnostics & Advanced Technology, Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management, China-Australia Joint Laboratory for Animal Health Big Data Analytics, College of Animal Science and Technology & College of Veterinary Medicine of Zhejiang A&F University, 666 Wusu Street, Lin'an District, Hangzhou, Zhejiang Province 311300, ChinaKey Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, Zhejiang Provincial Engineering Research Center for Animal Health Diagnostics & Advanced Technology, Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management, China-Australia Joint Laboratory for Animal Health Big Data Analytics, College of Animal Science and Technology & College of Veterinary Medicine of Zhejiang A&F University, 666 Wusu Street, Lin'an District, Hangzhou, Zhejiang Province 311300, ChinaKey Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, Zhejiang Provincial Engineering Research Center for Animal Health Diagnostics & Advanced Technology, Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management, China-Australia Joint Laboratory for Animal Health Big Data Analytics, College of Animal Science and Technology & College of Veterinary Medicine of Zhejiang A&F University, 666 Wusu Street, Lin'an District, Hangzhou, Zhejiang Province 311300, ChinaKey Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, Zhejiang Provincial Engineering Research Center for Animal Health Diagnostics & Advanced Technology, Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management, China-Australia Joint Laboratory for Animal Health Big Data Analytics, College of Animal Science and Technology & College of Veterinary Medicine of Zhejiang A&F University, 666 Wusu Street, Lin'an District, Hangzhou, Zhejiang Province 311300, ChinaKey Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, Zhejiang Provincial Engineering Research Center for Animal Health Diagnostics & Advanced Technology, Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management, China-Australia Joint Laboratory for Animal Health Big Data Analytics, College of Animal Science and Technology & College of Veterinary Medicine of Zhejiang A&F University, 666 Wusu Street, Lin'an District, Hangzhou, Zhejiang Province 311300, ChinaCorresponding author.; Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, Zhejiang Provincial Engineering Research Center for Animal Health Diagnostics & Advanced Technology, Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management, China-Australia Joint Laboratory for Animal Health Big Data Analytics, College of Animal Science and Technology & College of Veterinary Medicine of Zhejiang A&F University, 666 Wusu Street, Lin'an District, Hangzhou, Zhejiang Province 311300, ChinaAvian metapneumovirus (aMPV) represents a significant threat to the poultry industry, exhibiting a high degree of genetic diversity. Of these, the aMPV types A (aMPV-A), B (aMPV-B) and C (aMPV-C) are frequently detected in Chinese waterfowl and live poultry markets. Therefore, the rapid and accurate identification of these subtypes is of paramount importance in order to halt the spread of the disease. In this study, we have developed a multiplex real-time PCR assay endowed with the capacity to simultaneously discriminate aMPV-A, aMPV-B, and aMPV-C. This method demonstrates remarkable specificity, selectively amplifying aMPV-A, aMPV-B, and aMPV-C without cross-reactivity with other common avian pathogens. Furthermore, this method exhibits high sensitivity, with a detection threshold of 8.5 × 102 copies/μL for aMPV-A, aMPV-B, and aMPV-C. Moreover, the assay demonstrates reproducibility, as evidenced by intra- and inter-assay variability, with a coefficient of variation between 0.21% and 1.91%. Additionally, the receiver operating characteristic (ROC) curve analysis demonstrated that the multiplex real-time PCR assay exhibited high specificity and sensitivity (100.0% and 100.0% for aMPV-A, 90.9% and 100.0% for aMPV-B, 100% and 96.8% for aMPV-C) when compared with the classical aMPV real-time RT-PCR. Analyses of field samples (n=105) using the multiplex real-time PCR assay indicated that 35.2% (37/105) of samples were positive for aMPV, of which 29.7% (11/37) for aMPV-A, 32.4% (12/37) for aMPV-B and 37.8% (14/37) for aMPV-C. These data demonstrated that the multiplex real-time PCR assay can be used for epidemiological investigations of tree subtypes of aMPV and that aMPV had been observed to exhibit a proclivity for multiple types of co-infection in the Zhejiang province of China.http://www.sciencedirect.com/science/article/pii/S0032579124011866Avian metapneumovirusMultiplex real-time PCR assayReceiver operating characteristic |
| spellingShingle | Mingjun Su Jiongze Cheng Xiangwen Xu Yijia Liu Yulin Zhao Yutao Wang Xiaoxu Du Jiale Ying Junfang Yan Huihua Zheng Changyong Cheng Jing Sun Establishment and application of a one-step multiplex real-time PCR assay for detection of A, B, and C subtypes of avian metapneumovirus Poultry Science Avian metapneumovirus Multiplex real-time PCR assay Receiver operating characteristic |
| title | Establishment and application of a one-step multiplex real-time PCR assay for detection of A, B, and C subtypes of avian metapneumovirus |
| title_full | Establishment and application of a one-step multiplex real-time PCR assay for detection of A, B, and C subtypes of avian metapneumovirus |
| title_fullStr | Establishment and application of a one-step multiplex real-time PCR assay for detection of A, B, and C subtypes of avian metapneumovirus |
| title_full_unstemmed | Establishment and application of a one-step multiplex real-time PCR assay for detection of A, B, and C subtypes of avian metapneumovirus |
| title_short | Establishment and application of a one-step multiplex real-time PCR assay for detection of A, B, and C subtypes of avian metapneumovirus |
| title_sort | establishment and application of a one step multiplex real time pcr assay for detection of a b and c subtypes of avian metapneumovirus |
| topic | Avian metapneumovirus Multiplex real-time PCR assay Receiver operating characteristic |
| url | http://www.sciencedirect.com/science/article/pii/S0032579124011866 |
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