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Peer-reviewedCC BYBrazilian journal of microbiology : [publication of the Brazilian Society for Microbiology]

An endpoint multiplex PCR/RT-PCR for detection of viral agents of bovine respiratory disease.

  • Natália Hettwer Pedroso Alessio1,2
  • José Valter Joaquim Silva Júnior3,4,5,6,7
  • Iasmim Flôres Dionisio3,8
  • Vinicius Soares Dos Santos3
  • Viviani Gomes9
  • Rudi Weiblen3
  • Eduardo Furtado Flores3
9 affiliations
  1. 1Setor de Virologia, Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, Av. Roraima, 1000, Prédio 63 A, Camobi, Santa Maria, Rio Grande do Sul, 97105-900, Brazil. [email protected].
  2. 2Programa de Pós-graduação em Medicina Veterinária, Universidade Federal de Santa Maria, Santa Maria, Rio Grande do Sul, Brazil. [email protected].
  3. 3Setor de Virologia, Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, Av. Roraima, 1000, Prédio 63 A, Camobi, Santa Maria, Rio Grande do Sul, 97105-900, Brazil.
  4. 4Departamento de Microbiologia e Parasitologia, Universidade Federal de Santa Maria, Santa Maria, Rio Grande do Sul, Brazil.
  5. 5Laboratório NB3 de Neuroimunologia, Universidade Federal de Santa Maria, Santa Maria, Rio Grande do Sul, Brazil.
  6. 6Programa de Pós-graduação em Farmacologia, Universidade Federal de Santa Maria, Santa Maria, Rio Grande do Sul, Brazil.
  7. 7Setor de Virologia, Instituto Keizo Asami, Universidade Federal de Pernambuco, Pernambuco, Brazil.
  8. 8Programa de Pós-graduação em Medicina Veterinária, Universidade Federal de Santa Maria, Santa Maria, Rio Grande do Sul, Brazil.
  9. 9Departamento de Clínica Médica, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, Brasil.
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Abstract

Bovine respiratory disease (BRD) is a clinicopathological syndrome caused by complex interactions among host and environmental factors, viruses and bacteria. The multi-agent BRD etiology makes laboratory diagnosis challenging, as various pathogens should be investigated for an accurate results. Herein, we describe an endpoint multiplex PCR/RT-PCR for detection of major BRD-associated viruses: bovine herpesvirus 1 (BoAHV-1), bovine pestiviruses (BVDV-1, BVDV-2, HoBiPeV), bovine parainfluenza virus 3 (BPIV-3), bovine respiratory syncytial virus (BRSV) and bovine coronavirus (BCoV). We designed high-coverage and target-specific primers to be properly combined in the multiplex assay. Following standardization of individual reactions, the multiplex PCR/RT-PCR was optimized for reaction conditions and mix composition. The amplicons of the five targets were easily differentiated in agarose gel: 121 bp (BPIV-3), 157 bp (BoAHV-1), 204 bp (pestiviruses), 282 bp (BRSV) and 323 bp (BCoV). The limits of detection (LoDs) of our multiplex were 38 TCID₅₀ (BoAHV-1 and BPIV-3), 16 TCID₅₀ (pestiviruses), 13 TCID₅₀ (BRSV) and a 10- 2 dilution of a pool of five BCoV positive samples. No amplification of the main bacteria (Pasteurella multocida; Mannheimia haemolytica and Histophilus somni) potentially involved in BRD was observed. Finally, 289 respiratory specimens from animals with respiratory/nasal signs were evaluated by individual and multiplex PCR/RT-PCR tests to assess the diagnostic performance of the assay. Both tests resulted in 14.8% positive samples (43/289) being 19 for BPIV-3 (19/43), ten for BCoV (10/43), seven for BRSV (7/43), six for BoAHV-1 (6/43) and one for pestiviruses (1/43). Overall, our multiplex PCR/RT-PCR assay represents an alternative for an easier, faster and more practical laboratory diagnosis of viruses associated with BRD.

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Cite

BibTeXRIS
BibTeX
@article{alessio2026endpoint,
  title = {{An endpoint multiplex PCR/RT-PCR for detection of viral agents of bovine respiratory disease.}},
  author = {Natália Hettwer Pedroso Alessio and José Valter Joaquim Silva Júnior and Iasmim Flôres Dionisio and Vinicius Soares Dos Santos and Viviani Gomes and Rudi Weiblen and Eduardo Furtado Flores},
  journal = {Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology]},
  year = {2026},
  month = nov,
  doi = {10.1007/s42770-026-02097-2},
  url = {https://europepmc.org/article/MED/42970197},
  note = {CC BY},
}
RIS
TY  - JOUR
TI  - An endpoint multiplex PCR/RT-PCR for detection of viral agents of bovine respiratory disease.
AU  - Natália Hettwer Pedroso Alessio
AU  - José Valter Joaquim Silva Júnior
AU  - Iasmim Flôres Dionisio
AU  - Vinicius Soares Dos Santos
AU  - Viviani Gomes
AU  - Rudi Weiblen
AU  - Eduardo Furtado Flores
PY  - 2026
DA  - 2026/11/02
JO  - Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology]
DO  - 10.1007/s42770-026-02097-2
UR  - https://europepmc.org/article/MED/42970197
AB  - Bovine respiratory disease (BRD) is a clinicopathological syndrome caused by complex interactions among host and environmental factors, viruses and bacteria. The multi-agent BRD etiology makes laboratory diagnosis challenging, as various pathogens should be investigated for an accurate results. Herein, we describe an endpoint multiplex PCR/RT-PCR for detection of major BRD-associated viruses: bovine herpesvirus 1 (BoAHV-1), bovine pestiviruses (BVDV-1, BVDV-2, HoBiPeV), bovine parainfluenza virus 3 (BPIV-3), bovine respiratory syncytial virus (BRSV) and bovine coronavirus (BCoV). We designed high-coverage and target-specific primers to be properly combined in the multiplex assay. Following standardization of individual reactions, the multiplex PCR/RT-PCR was optimized for reaction conditions and mix composition. The amplicons of the five targets were easily differentiated in agarose gel: 121 bp (BPIV-3), 157 bp (BoAHV-1), 204 bp (pestiviruses), 282 bp (BRSV) and 323 bp (BCoV). The limits of detection (LoDs) of our multiplex were 38 TCID₅₀ (BoAHV-1 and BPIV-3), 16 TCID₅₀ (pestiviruses), 13 TCID₅₀ (BRSV) and a 10- 2 dilution of a pool of five BCoV positive samples. No amplification of the main bacteria (Pasteurella multocida; Mannheimia haemolytica and Histophilus somni) potentially involved in BRD was observed. Finally, 289 respiratory specimens from animals with respiratory/nasal signs were evaluated by individual and multiplex PCR/RT-PCR tests to assess the diagnostic performance of the assay. Both tests resulted in 14.8% positive samples (43/289) being 19 for BPIV-3 (19/43), ten for BCoV (10/43), seven for BRSV (7/43), six for BoAHV-1 (6/43) and one for pestiviruses (1/43). Overall, our multiplex PCR/RT-PCR assay represents an alternative for an easier, faster and more practical laboratory diagnosis of viruses associated with BRD.
SN  - 1517-8382
SN  - 1678-4405
ER  -