PCR NESTED Multiplex: detecção de Orthopoxvirus e Parapoxvirus diretamente de amostras clínicas

Abstract

The Poxviridae family contains the largest and most complex animal viruses capable of infecting vertebrate and invertebrate hosts, it is divided into two wide subfamilies Chordopoxvirinae and Entomopoxvirinae that infect vertebrate and invertebrate hosts, respectively. Only members of the Chordopoxvirinae subfamily are capable of infecting vertebrate host. The Orthopoxvirus (OPV) and Parapoxvirus (PPV), genera of this subfamily, have been associated with exanthema outbreaks worldwide. Some species of these genera are capable of infecting humans and domestic animals, causing serious economic losses and public health impact. Fast and efficient methods with high specificity are required to detect and epidemiologically monitor such poxviruses. At the present work is described the development of a multiplex nested PCR which detects both types of PPV and OPV rashes injuries directly, without prior viral DNA extraction or virus isolation. Based on the evaluation and combination of sets of published primers the multiplex nested PCR for OPV / PPV was developed and applied to the detection of target pathogens. The method showed high sensitivity and sequencing of the amplified confirmed the specificity. The multiplex nested PCR for OPV / PPV were used for analysis of rash injuries samples collected during outbreaks of bovine vaccinia (BV) and contagious ecthyma (CE), confirming its applicability. These results suggest that the presented multiplex PCR provides a high robustness and high sensitivity method for detecting OPV and PPV directly from clinical specimens. The method can be used for virus identification and monitoring, especially in areas where there is PPV and OPV at the same time.