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Open Access Research

Nested-multiplex PCR detection of Orthopoxvirus and Parapoxvirus directly from exanthematic clinical samples

Jônatas S Abrahão1, Larissa S Lima1, Felipe L Assis1, Pedro A Alves1, André T Silva-Fernandes1, Marcela MG Cota1, Vanessa M Ferreira1, Rafael K Campos1, Carlos Mazur3, Zélia IP Lobato2, Giliane S Trindade1 and Erna G Kroon1*

Author Affiliations

1 Laboratório de Vírus, Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais. Av. Antônio Carlos, 6627, caixa postal 486, CEP: 31270-901, Belo Horizonte, MG, Brazil

2 Departamento de Medicina Veterinária Preventiva, Escola de Veterinária, Universidade Federal de Minas Gerais. Av. Antônio Carlos, 6627, CEP: 31270-901, Belo Horizonte, MG, Brazil

3 Departamento de Microbiologia e Imunologia Veterinária, Universidade Federal Rural do Rio de Janeiro. BR465, Km07, Boa Esperança. CEP: 23890-000, Seropedica, Rio de Janeiro, Brazil

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Virology Journal 2009, 6:140  doi:10.1186/1743-422X-6-140

Published: 11 September 2009

Abstract

Background

Orthopoxvirus (OPV) and Parapoxvirus (PPV) have been associated with worldwide exanthematic outbreaks. Some species of these genera are able to infect humans and domestic animals, causing serious economic losses and public health impact. Rapid, useful and highly specific methods are required to detect and epidemiologically monitor such poxviruses. In the present paper, we describe the development of a nested-multiplex PCR method for the simultaneous detection of OPV and PPV species directly from exanthematic lesions, with no previous viral isolation or DNA extraction.

Methods and Results

The OPV/PPV nested-multiplex PCR was developed based on the evaluation and combination of published primer sets, and was applied to the detection of the target pathogens. The method showed high sensitivity, and the specificity was confirmed by amplicon sequencing. Exanthematic lesion samples collected during bovine vaccinia or contagious ecthyma outbreaks were submitted to OPV/PPV nested-multiplex PCR and confirmed its applicability.

Conclusion

These results suggest that the presented multiplex PCR provides a highly robust and sensitive method to detect OPV and PPV directly from clinical samples. The method can be used for viral identification and monitoring, especially in areas where OPV and PPV co-circulate.