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


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.


CONCLUSIONS
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.
Abrahão
Jônatas S
JS
Departamento de Microbiologia, Laboratório de Vírus, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, CEP: 31270-901 Belo Horizonte, MG, Brazil. jonatas.abrahao@gmail.com
Lima
Larissa S
LS
Assis
Felipe L
FL
Alves
Pedro A
PA
Silva-Fernandes
André T
AT
Cota
Marcela M G
MM
Ferreira
Vanessa M
VM
Campos
Rafael K
RK
Mazur
Carlos
C
Lobato
Zélia I P
ZI
Trindade
Giliane S
GS
Kroon
Erna G
EG
D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000821:virology; D017931:DNA Primers / Q000235:genetics; D015511:Goat Diseases / Q000821:virology; D006041:Goats; D006801:Humans; D008969:Molecular Sequence Data; D018155:Orthopoxvirus / Q000235:genetics / Q000302:isolation & purification*; D018157:Parapoxvirus / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011213:Poxviridae Infections / Q000662:veterinary* / Q000821:virology*; D012756:Sheep; D012757:Sheep Diseases / Q000821:virology
D017931:DNA Primers