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| [PMID]: | 27793645 |
| [Au] Autor: | Cargnelutti JF; Weiblen R; Flores EF |
| [Ad] Endereço: | Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, Av. Roraima, 1000, building 63A, Santa Maria, Rio Grande do Sul, zip-code 97105-900, Brazil. |
| [Ti] Título: | A multiplex PCR for viruses associated with exanthematic and vesicular disease in cattle. |
| [So] Source: | J Virol Methods;239:38-41, 2017 Jan. | | [Is] ISSN: | 1879-0984 |
| [Cp] País de publicação: | Netherlands |
| [La] Idioma: | eng |
| [Ab] Resumo: | Exanthematic and papulo-vesicular lesions in the udder and teats of milking cows are fairly common in some Brazilian dairies, especially those with poor sanitary conditions and hand milking. The orthopoxvirus Vaccinia virus (VACV) and the parapoxviruses Pseudocowpox virus (PCPV) and Bovine popular stomatitis virus (BPSV) have been frequently associated with such conditions. Elsewhere, Bovine herpesvirus 2 (BoHV-2) has also been associated with similar clinical signs. Thus, we herein describe a conventional multiplex PCR designed to detect the genome of these viruses in clinical samples while differentiating among them by amplicon size. For this, primer sets targeting the orthopoxvirus vascular growth factor (amplicon size 292bp), PCPV (374bp) and BSPV (607bp) B2L genes, and the BoHV-2 DNA polymerase gene (138bp) were selected. The chosen primers anneal within the same temperature range and do not interfere with each other during the PCR amplification. PCR conditions were initially standardized for each agent in individual PCR reactions firstly using the target virus as positive control followed by using a mixture of all four virues. Lastly, a multiplex PCR containing the four sets of primers was set up to amplify all four targeted viruses in one reaction. The multiplex PCR was able to detect DNA extracted from cell culture supernatants containing 20 TCID of BoHV-2 and 50 TCID of VACV. Further, the test could detect the viral genomes in 1:10, 1:50 and 1:1000 dilutions of total DNA extracted from clinical specimens (e.g. scabs, crusts) of natural cases (PCPV, VACV and BPSV) and 1:10 dilutions of DNA extracted from scabs collected from BoHV-2 experimentally infected cattle. A possible amplification of other orthopoxviruses, predicted by in silico analysis, was considered to not represent an important pitfall since these are exotic in Brazil, very rare, or viruses not associated with cattle. For definitive agent identification amplicon sequencing needs to be conducted. Thus, this multiplex PCR seems suitable for initial detection and identification of the agents involved in exanthematic and vesicular disease, providing a sensitive and specific diagnosis for such conditions in dairy cows. |
| [Mh] Termos MeSH primário: |
Doenças dos Bovinos/diagnóstico
Doenças dos Bovinos/virologia
Reação em Cadeia da Polimerase Multiplex/métodos
Infecções por Poxviridae/veterinária
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| [Mh] Termos MeSH secundário: |
Animais
Brasil
Bovinos
Primers do DNA
DNA Viral/genética
Genes Virais
Genoma Viral
Orthopoxvirus/genética
Orthopoxvirus/isolamento & purificação
Parapoxvirus/genética
Parapoxvirus/isolamento & purificação
Infecções por Poxviridae/diagnóstico
Infecções por Poxviridae/virologia
Vírus da Pseudovaríola das Vacas/genética
Vírus da Pseudovaríola das Vacas/isolamento & purificação
Vírus Vaccinia/genética
Vírus Vaccinia/isolamento & purificação
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| [Pt] Tipo de publicação: | JOURNAL ARTICLE |
[Nm] Nome de substância:
| 0 (DNA Primers); 0 (DNA, Viral) |
| [Em] Mês de entrada: | 1710 |
| [Cu] Atualização por classe: | 171016 |
[Lr] Data última revisão:
| 171016 |
| [Sb] Subgrupo de revista: | IM |
| [Da] Data de entrada para processamento: | 161030 |
| [St] Status: | MEDLINE |
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