(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.