Development of a Multiplex Real-Time PCR Assay for the Detection of Eight Pathogens Associated with Bovine Respiratory Disease Complex from Clinical Samples
Bovine respiratory disease complex (BRDC) is one of the primary causes of morbidity, mortality, and economic loss in cattle worldwide. Accurate and rapid identification of causative pathogenic agents is essential for effective disease management and control. In this study, a novel multiplex fluoresc...
Saved in:
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2025-07-01
|
| Series: | Microorganisms |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2076-2607/13/7/1629 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1849252029853073408 |
|---|---|
| author | Fuxing Hao Chunhao Tao Ruilong Xiao Ying Huang Weifeng Yuan Zhen Wang Hong Jia |
| author_facet | Fuxing Hao Chunhao Tao Ruilong Xiao Ying Huang Weifeng Yuan Zhen Wang Hong Jia |
| author_sort | Fuxing Hao |
| collection | DOAJ |
| description | Bovine respiratory disease complex (BRDC) is one of the primary causes of morbidity, mortality, and economic loss in cattle worldwide. Accurate and rapid identification of causative pathogenic agents is essential for effective disease management and control. In this study, a novel multiplex fluorescence-based quantitative polymerase chain reaction (qPCR) assay was developed for the simultaneous detection of eight major pathogens associated with BRDC. The targeted pathogens included the following: bovine viral diarrhea virus (BVDV), bovine parainfluenza virus type 3 (BPIV3), bovine respiratory syncytial virus (BRSV), bovine coronavirus (BcoV), <i>Mycoplasma bovis</i> (M.bovis), <i>Pasteurella multocida</i> (PM), <i>Mannheimia haemolytica</i> (MH), and infectious bovine rhinotracheitis virus (IBRV). The assay was rigorously optimized to ensure high specificity with no cross-reactivity among targets. The limit of detection (LOD) was determined to be as low as 5 copies per reaction for all target pathogens. The coefficient of variation (CVs) for both intra-assay and inter-assay measurements were consistently below 2%, demonstrating excellent reproducibility. To validate the clinical utility of the assay, a total of 1012 field samples were tested, including 504 nasal swabs from Farm A and 508 from Farm B in Jiangsu Province. BVDV, BcoV, PM, and MH were detected from Farm A, with a BVDV-positive rate of 21.63% (109/504), BcoV-positive rate of 26.79% (135/504), PM-positive rate of 28.77% (145/504), and MH-positive rate of 15.08% (76/504). Also, BcoV, PM, MH, and IBRV were detected from Farm B, with a BcoV-positive rate of 2.36% (12/508), PM-positive rate of 1.38% (7/508), MH-positive rate of 14.76% (75/508), and IBRV-positive rate of 5.51% (28/508). Notably, a significant proportion of samples showed evidence of mixed infections, underscoring the complexity of BRDC etiology and the importance of a multiplex diagnostic approach. In conclusion, the developed multiplex qPCR assay provides a reliable, rapid, and cost-effective tool for simultaneous detection of multiple BRDC-associated pathogens, which will hold great promise for enhancing disease surveillance, early diagnosis, and targeted intervention strategies, ultimately contributing to improved BRDC management and cattle health outcomes. |
| format | Article |
| id | doaj-art-ce8ddd28c89f4f66a5db6934db9d0b72 |
| institution | Kabale University |
| issn | 2076-2607 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Microorganisms |
| spelling | doaj-art-ce8ddd28c89f4f66a5db6934db9d0b722025-08-20T03:56:45ZengMDPI AGMicroorganisms2076-26072025-07-01137162910.3390/microorganisms13071629Development of a Multiplex Real-Time PCR Assay for the Detection of Eight Pathogens Associated with Bovine Respiratory Disease Complex from Clinical SamplesFuxing Hao0Chunhao Tao1Ruilong Xiao2Ying Huang3Weifeng Yuan4Zhen Wang5Hong Jia6Jiangsu Agri-Animal Husbandry Vocational College, Taizhou 225300, ChinaInstitute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, ChinaInstitute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, ChinaInstitute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, ChinaInstitute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, ChinaInstitute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, ChinaInstitute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, ChinaBovine respiratory disease complex (BRDC) is one of the primary causes of morbidity, mortality, and economic loss in cattle worldwide. Accurate and rapid identification of causative pathogenic agents is essential for effective disease management and control. In this study, a novel multiplex fluorescence-based quantitative polymerase chain reaction (qPCR) assay was developed for the simultaneous detection of eight major pathogens associated with BRDC. The targeted pathogens included the following: bovine viral diarrhea virus (BVDV), bovine parainfluenza virus type 3 (BPIV3), bovine respiratory syncytial virus (BRSV), bovine coronavirus (BcoV), <i>Mycoplasma bovis</i> (M.bovis), <i>Pasteurella multocida</i> (PM), <i>Mannheimia haemolytica</i> (MH), and infectious bovine rhinotracheitis virus (IBRV). The assay was rigorously optimized to ensure high specificity with no cross-reactivity among targets. The limit of detection (LOD) was determined to be as low as 5 copies per reaction for all target pathogens. The coefficient of variation (CVs) for both intra-assay and inter-assay measurements were consistently below 2%, demonstrating excellent reproducibility. To validate the clinical utility of the assay, a total of 1012 field samples were tested, including 504 nasal swabs from Farm A and 508 from Farm B in Jiangsu Province. BVDV, BcoV, PM, and MH were detected from Farm A, with a BVDV-positive rate of 21.63% (109/504), BcoV-positive rate of 26.79% (135/504), PM-positive rate of 28.77% (145/504), and MH-positive rate of 15.08% (76/504). Also, BcoV, PM, MH, and IBRV were detected from Farm B, with a BcoV-positive rate of 2.36% (12/508), PM-positive rate of 1.38% (7/508), MH-positive rate of 14.76% (75/508), and IBRV-positive rate of 5.51% (28/508). Notably, a significant proportion of samples showed evidence of mixed infections, underscoring the complexity of BRDC etiology and the importance of a multiplex diagnostic approach. In conclusion, the developed multiplex qPCR assay provides a reliable, rapid, and cost-effective tool for simultaneous detection of multiple BRDC-associated pathogens, which will hold great promise for enhancing disease surveillance, early diagnosis, and targeted intervention strategies, ultimately contributing to improved BRDC management and cattle health outcomes.https://www.mdpi.com/2076-2607/13/7/1629bovine respiratory disease complex (BRDC)multiplex qPCRdiagnosismixed infection |
| spellingShingle | Fuxing Hao Chunhao Tao Ruilong Xiao Ying Huang Weifeng Yuan Zhen Wang Hong Jia Development of a Multiplex Real-Time PCR Assay for the Detection of Eight Pathogens Associated with Bovine Respiratory Disease Complex from Clinical Samples Microorganisms bovine respiratory disease complex (BRDC) multiplex qPCR diagnosis mixed infection |
| title | Development of a Multiplex Real-Time PCR Assay for the Detection of Eight Pathogens Associated with Bovine Respiratory Disease Complex from Clinical Samples |
| title_full | Development of a Multiplex Real-Time PCR Assay for the Detection of Eight Pathogens Associated with Bovine Respiratory Disease Complex from Clinical Samples |
| title_fullStr | Development of a Multiplex Real-Time PCR Assay for the Detection of Eight Pathogens Associated with Bovine Respiratory Disease Complex from Clinical Samples |
| title_full_unstemmed | Development of a Multiplex Real-Time PCR Assay for the Detection of Eight Pathogens Associated with Bovine Respiratory Disease Complex from Clinical Samples |
| title_short | Development of a Multiplex Real-Time PCR Assay for the Detection of Eight Pathogens Associated with Bovine Respiratory Disease Complex from Clinical Samples |
| title_sort | development of a multiplex real time pcr assay for the detection of eight pathogens associated with bovine respiratory disease complex from clinical samples |
| topic | bovine respiratory disease complex (BRDC) multiplex qPCR diagnosis mixed infection |
| url | https://www.mdpi.com/2076-2607/13/7/1629 |
| work_keys_str_mv | AT fuxinghao developmentofamultiplexrealtimepcrassayforthedetectionofeightpathogensassociatedwithbovinerespiratorydiseasecomplexfromclinicalsamples AT chunhaotao developmentofamultiplexrealtimepcrassayforthedetectionofeightpathogensassociatedwithbovinerespiratorydiseasecomplexfromclinicalsamples AT ruilongxiao developmentofamultiplexrealtimepcrassayforthedetectionofeightpathogensassociatedwithbovinerespiratorydiseasecomplexfromclinicalsamples AT yinghuang developmentofamultiplexrealtimepcrassayforthedetectionofeightpathogensassociatedwithbovinerespiratorydiseasecomplexfromclinicalsamples AT weifengyuan developmentofamultiplexrealtimepcrassayforthedetectionofeightpathogensassociatedwithbovinerespiratorydiseasecomplexfromclinicalsamples AT zhenwang developmentofamultiplexrealtimepcrassayforthedetectionofeightpathogensassociatedwithbovinerespiratorydiseasecomplexfromclinicalsamples AT hongjia developmentofamultiplexrealtimepcrassayforthedetectionofeightpathogensassociatedwithbovinerespiratorydiseasecomplexfromclinicalsamples |