A multiplex TaqMan real-time RT-PCR assay for the simultaneous detection of H4, H6, and H10 avian influenza viruses
Avian influenza viruses (AIVs) have caused a large number of epidemics in domestic and wild birds, and even posed a health challenge to humans. Highly pathogenic AIVs have attracted the most public attention. However, low pathogenic AIVs, including H4, H6, and H10 subtype AIVs, have spread covertly...
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Elsevier
2023-05-01
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| Series: | Heliyon |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2405844023028542 |
| _version_ | 1797815648433209344 |
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| author | Fan Yang Sijing Yan Linwei Zhu Hangping Yao Dalu Dong Danna Wu Nanping Wu Chunsheng Ye Haibo Wu |
| author_facet | Fan Yang Sijing Yan Linwei Zhu Hangping Yao Dalu Dong Danna Wu Nanping Wu Chunsheng Ye Haibo Wu |
| author_sort | Fan Yang |
| collection | DOAJ |
| description | Avian influenza viruses (AIVs) have caused a large number of epidemics in domestic and wild birds, and even posed a health challenge to humans. Highly pathogenic AIVs have attracted the most public attention. However, low pathogenic AIVs, including H4, H6, and H10 subtype AIVs, have spread covertly in domestic poultry, without obvious clinical signs. The emergence of human infections with H6 and H10 AIVs and the evidence of seropositivity of H4 AIV in poultry-exposed individuals indicated that these AIVs sporadically infect humans and could cause a potential pandemic. Therefore, a rapid and sensitive diagnostic method to simultaneously detect Eurasian lineage H4, H6, and H10 subtype AIVs is urgently required. Four singleplex real-time RT-PCR (RRT-PCR) assays were established based on carefully designed primers and probes of the conserved regions of the matrix, H4, H6, and H10 genes and combined into a multiplex RRT-PCR method to simultaneously detect H4, H6, and H10 AIVs in one reaction. The detection limit of the multiplex RRT-PCR method was 1–10 copies per reaction when detecting standard plasmids, and showed no cross-reaction against other subtype AIVs and other common avian viruses. Additionally, this method was suitable to detect the AIVs in samples from different sources, the results of which showed high consistency with virus isolation and a commercial influenza detection kit. In summary, this rapid, convenient, and practical multiplex RRT-PCR method could be applied in laboratory testing and clinical screening to detect AIVs. |
| first_indexed | 2024-03-13T08:25:58Z |
| format | Article |
| id | doaj.art-a1b0619be885431ab2294aa5696f1cd5 |
| institution | Directory Open Access Journal |
| issn | 2405-8440 |
| language | English |
| last_indexed | 2024-03-13T08:25:58Z |
| publishDate | 2023-05-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Heliyon |
| spelling | doaj.art-a1b0619be885431ab2294aa5696f1cd52023-05-31T04:45:02ZengElsevierHeliyon2405-84402023-05-0195e15647A multiplex TaqMan real-time RT-PCR assay for the simultaneous detection of H4, H6, and H10 avian influenza virusesFan Yang0Sijing Yan1Linwei Zhu2Hangping Yao3Dalu Dong4Danna Wu5Nanping Wu6Chunsheng Ye7Haibo Wu8State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310003, ChinaState Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310003, ChinaState Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310003, ChinaState Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310003, China; Jinan Microecological Biomedicine Shandong Laboratory, Jinan, 250117, ChinaHangzhou Biotest Biotech Co., Ltd., 27 Tuyi Road, Cangqian Street, Yuhang District, Hangzhou, 311121, ChinaHangzhou Biotest Biotech Co., Ltd., 27 Tuyi Road, Cangqian Street, Yuhang District, Hangzhou, 311121, ChinaState Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310003, China; Jinan Microecological Biomedicine Shandong Laboratory, Jinan, 250117, ChinaHangzhou Biotest Biotech Co., Ltd., 27 Tuyi Road, Cangqian Street, Yuhang District, Hangzhou, 311121, China; Corresponding author. Hangzhou Biotest Biotech Co., Ltd., 27 Tuyi Road, Cangqian Street, Yuhang District, Hangzhou 311121, Zhejiang, China.State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310003, China; Jinan Microecological Biomedicine Shandong Laboratory, Jinan, 250117, China; Corresponding author. State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, the First Affiliated Hospital, School of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou, 310003, Zhejiang, China.Avian influenza viruses (AIVs) have caused a large number of epidemics in domestic and wild birds, and even posed a health challenge to humans. Highly pathogenic AIVs have attracted the most public attention. However, low pathogenic AIVs, including H4, H6, and H10 subtype AIVs, have spread covertly in domestic poultry, without obvious clinical signs. The emergence of human infections with H6 and H10 AIVs and the evidence of seropositivity of H4 AIV in poultry-exposed individuals indicated that these AIVs sporadically infect humans and could cause a potential pandemic. Therefore, a rapid and sensitive diagnostic method to simultaneously detect Eurasian lineage H4, H6, and H10 subtype AIVs is urgently required. Four singleplex real-time RT-PCR (RRT-PCR) assays were established based on carefully designed primers and probes of the conserved regions of the matrix, H4, H6, and H10 genes and combined into a multiplex RRT-PCR method to simultaneously detect H4, H6, and H10 AIVs in one reaction. The detection limit of the multiplex RRT-PCR method was 1–10 copies per reaction when detecting standard plasmids, and showed no cross-reaction against other subtype AIVs and other common avian viruses. Additionally, this method was suitable to detect the AIVs in samples from different sources, the results of which showed high consistency with virus isolation and a commercial influenza detection kit. In summary, this rapid, convenient, and practical multiplex RRT-PCR method could be applied in laboratory testing and clinical screening to detect AIVs.http://www.sciencedirect.com/science/article/pii/S2405844023028542Avian influenza virusMultiplex real-time RT-PCRSubtypingVirus diagnosis |
| spellingShingle | Fan Yang Sijing Yan Linwei Zhu Hangping Yao Dalu Dong Danna Wu Nanping Wu Chunsheng Ye Haibo Wu A multiplex TaqMan real-time RT-PCR assay for the simultaneous detection of H4, H6, and H10 avian influenza viruses Heliyon Avian influenza virus Multiplex real-time RT-PCR Subtyping Virus diagnosis |
| title | A multiplex TaqMan real-time RT-PCR assay for the simultaneous detection of H4, H6, and H10 avian influenza viruses |
| title_full | A multiplex TaqMan real-time RT-PCR assay for the simultaneous detection of H4, H6, and H10 avian influenza viruses |
| title_fullStr | A multiplex TaqMan real-time RT-PCR assay for the simultaneous detection of H4, H6, and H10 avian influenza viruses |
| title_full_unstemmed | A multiplex TaqMan real-time RT-PCR assay for the simultaneous detection of H4, H6, and H10 avian influenza viruses |
| title_short | A multiplex TaqMan real-time RT-PCR assay for the simultaneous detection of H4, H6, and H10 avian influenza viruses |
| title_sort | multiplex taqman real time rt pcr assay for the simultaneous detection of h4 h6 and h10 avian influenza viruses |
| topic | Avian influenza virus Multiplex real-time RT-PCR Subtyping Virus diagnosis |
| url | http://www.sciencedirect.com/science/article/pii/S2405844023028542 |
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