The quadruplex fluorescent quantitative PCR method for the simultaneous detection of respiratory diseases in quail: Pasteurella multocida, Avibacterium paragallinarum, Mycoplasma gallisepticum, and Mycoplasma synoviae
BackgroundThe quail farming industry constitutes an important component of China’s agricultural sector. However, it is frequently threatened by various bacterial and mycoplasmal infections, particularly respiratory diseases caused by Pasteurella multocida, Avibacterium paragallinarum, Mycoplasma gal...
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Frontiers Media S.A.
2025-06-01
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| author | Haojie Wang Lihong Xue Longxi Wang Yixuan Liu Jianxing Chen Yue Sun Tongqing An Changwen Li Hongyan Chen Changqing Yu Changyou Xia He Zhang |
| author_facet | Haojie Wang Lihong Xue Longxi Wang Yixuan Liu Jianxing Chen Yue Sun Tongqing An Changwen Li Hongyan Chen Changqing Yu Changyou Xia He Zhang |
| author_sort | Haojie Wang |
| collection | DOAJ |
| description | BackgroundThe quail farming industry constitutes an important component of China’s agricultural sector. However, it is frequently threatened by various bacterial and mycoplasmal infections, particularly respiratory diseases caused by Pasteurella multocida, Avibacterium paragallinarum, Mycoplasma gallisepticum, and Mycoplasma synoviae. These pathogens commonly result in co-infections or secondary infections, and their clinical presentations are often indistinguishable due to the similarity of symptoms.MethodsFour sets of primers and probes were designed based on the GenBank-registered gene sequences: the kmt1 gene of P. multocida, the recN gene of A. paragallinarum, the mgc2 gene of M. gallisepticum, and the vlhA gene of M. synoviae. Reaction conditions were optimized accordingly. A recombinant plasmid standard was constructed for the generation of standard curves. The sensitivity, specificity, reproducibility, and accuracy of the assay were systematically evaluated.ResultsThe constructed standard curves demonstrated strong linearity (R2 = 1.000, 0.998, 1.000, and 1.000), with high amplification efficiencies (107.09, 91.23, 112.10, and 125.51%, respectively). The detection limit for each recombinant plasmid standard was as low as 10 copies. No cross-reactivity was observed with non-target pathogens, including avian pox virus, Escherichia coli, Salmonella spp., Newcastle disease virus, infectious bronchitis virus, infectious laryngotracheitis virus, and Staphylococcus aureus. The assay exhibited excellent reproducibility, with inter- and intra-assay coefficient of variation (CV) values ranging from 0.11 to 1.41%. Among 126 clinical samples, P. multocida was detected in 6 samples, A. paragallinarum in 3, M. gallisepticum in 6, and M. synoviae in 4. These results were consistent with those obtained using previously established methods.DiscussionA highly sensitive, specific, rapid, and efficient quadruplex fluorescence quantitative PCR assay was successfully developed for the simultaneous detection and identification of Pasteurella multocida, Avibacterium paragallinarum, Mycoplasma gallisepticum, and Mycoplasma synoviae. |
| format | Article |
| id | doaj-art-34eb95a0da524414baa722e0329c8bee |
| institution | Kabale University |
| issn | 1664-302X |
| language | English |
| publishDate | 2025-06-01 |
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| series | Frontiers in Microbiology |
| spelling | doaj-art-34eb95a0da524414baa722e0329c8bee2025-08-20T03:24:51ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2025-06-011610.3389/fmicb.2025.16053561605356The quadruplex fluorescent quantitative PCR method for the simultaneous detection of respiratory diseases in quail: Pasteurella multocida, Avibacterium paragallinarum, Mycoplasma gallisepticum, and Mycoplasma synoviaeHaojie Wang0Lihong Xue1Longxi Wang2Yixuan Liu3Jianxing Chen4Yue Sun5Tongqing An6Changwen Li7Hongyan Chen8Changqing Yu9Changyou Xia10He Zhang11State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaState Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaState Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaState Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaState Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaState Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaState Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaState Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaState Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaSchool of Advanced Agricultural Sciences, Yibin Vocational and Technical College, Yibin, ChinaState Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaState Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, ChinaBackgroundThe quail farming industry constitutes an important component of China’s agricultural sector. However, it is frequently threatened by various bacterial and mycoplasmal infections, particularly respiratory diseases caused by Pasteurella multocida, Avibacterium paragallinarum, Mycoplasma gallisepticum, and Mycoplasma synoviae. These pathogens commonly result in co-infections or secondary infections, and their clinical presentations are often indistinguishable due to the similarity of symptoms.MethodsFour sets of primers and probes were designed based on the GenBank-registered gene sequences: the kmt1 gene of P. multocida, the recN gene of A. paragallinarum, the mgc2 gene of M. gallisepticum, and the vlhA gene of M. synoviae. Reaction conditions were optimized accordingly. A recombinant plasmid standard was constructed for the generation of standard curves. The sensitivity, specificity, reproducibility, and accuracy of the assay were systematically evaluated.ResultsThe constructed standard curves demonstrated strong linearity (R2 = 1.000, 0.998, 1.000, and 1.000), with high amplification efficiencies (107.09, 91.23, 112.10, and 125.51%, respectively). The detection limit for each recombinant plasmid standard was as low as 10 copies. No cross-reactivity was observed with non-target pathogens, including avian pox virus, Escherichia coli, Salmonella spp., Newcastle disease virus, infectious bronchitis virus, infectious laryngotracheitis virus, and Staphylococcus aureus. The assay exhibited excellent reproducibility, with inter- and intra-assay coefficient of variation (CV) values ranging from 0.11 to 1.41%. Among 126 clinical samples, P. multocida was detected in 6 samples, A. paragallinarum in 3, M. gallisepticum in 6, and M. synoviae in 4. These results were consistent with those obtained using previously established methods.DiscussionA highly sensitive, specific, rapid, and efficient quadruplex fluorescence quantitative PCR assay was successfully developed for the simultaneous detection and identification of Pasteurella multocida, Avibacterium paragallinarum, Mycoplasma gallisepticum, and Mycoplasma synoviae.https://www.frontiersin.org/articles/10.3389/fmicb.2025.1605356/fullPasteurella multocidaAvibacterium paragallinarumMycoplasma gallisepticumMycoplasma synoviaequadruplex fluorescent |
| spellingShingle | Haojie Wang Lihong Xue Longxi Wang Yixuan Liu Jianxing Chen Yue Sun Tongqing An Changwen Li Hongyan Chen Changqing Yu Changyou Xia He Zhang The quadruplex fluorescent quantitative PCR method for the simultaneous detection of respiratory diseases in quail: Pasteurella multocida, Avibacterium paragallinarum, Mycoplasma gallisepticum, and Mycoplasma synoviae Frontiers in Microbiology Pasteurella multocida Avibacterium paragallinarum Mycoplasma gallisepticum Mycoplasma synoviae quadruplex fluorescent |
| title | The quadruplex fluorescent quantitative PCR method for the simultaneous detection of respiratory diseases in quail: Pasteurella multocida, Avibacterium paragallinarum, Mycoplasma gallisepticum, and Mycoplasma synoviae |
| title_full | The quadruplex fluorescent quantitative PCR method for the simultaneous detection of respiratory diseases in quail: Pasteurella multocida, Avibacterium paragallinarum, Mycoplasma gallisepticum, and Mycoplasma synoviae |
| title_fullStr | The quadruplex fluorescent quantitative PCR method for the simultaneous detection of respiratory diseases in quail: Pasteurella multocida, Avibacterium paragallinarum, Mycoplasma gallisepticum, and Mycoplasma synoviae |
| title_full_unstemmed | The quadruplex fluorescent quantitative PCR method for the simultaneous detection of respiratory diseases in quail: Pasteurella multocida, Avibacterium paragallinarum, Mycoplasma gallisepticum, and Mycoplasma synoviae |
| title_short | The quadruplex fluorescent quantitative PCR method for the simultaneous detection of respiratory diseases in quail: Pasteurella multocida, Avibacterium paragallinarum, Mycoplasma gallisepticum, and Mycoplasma synoviae |
| title_sort | quadruplex fluorescent quantitative pcr method for the simultaneous detection of respiratory diseases in quail pasteurella multocida avibacterium paragallinarum mycoplasma gallisepticum and mycoplasma synoviae |
| topic | Pasteurella multocida Avibacterium paragallinarum Mycoplasma gallisepticum Mycoplasma synoviae quadruplex fluorescent |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2025.1605356/full |
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