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

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.

Figure 1

(A) OPV/PPV nested-multiplex standardization and (B) sensitivity tests. Exanthematic lesions from BV and CE outbreaks were used in PCR standardization and sensitivity assays. Different thermal and chemical conditions were tested. (A) lane 1-3: BV scabs and vesicles presenting OPV vgf gene amplification (170 bp); lane 4-6: CE scabs presenting PPV b2l gene amplification (592 bp); lane 7: negative control; lane 8-9: BV and CE scabs, simulating a possible co-infection, presenting the simultaneous amplification of OPV vgf and PPV b2l genes. (B) PCR sensitivity tests performed with different concentrations of vgf or b2l fragments. The nested-multiplex was able to detect OPV and PPV DNA until reactions in which there was 1 ng of vgf or b2l genes. The PCR products were electrophoresed on 8% PAGE gels and silver stained. NC: negative control.