Novel and highly sensitive SYBR® Green real-time PCR for poxvirus detection in odontocete cetaceans
Sacristán, Carlos | Catão-Dias, J. L. | Ewbank, Ana Carolina | Ferreira-Machado, Eduardo | Neves, Elena | Santos-Neto, E. B. | Azevedo, A. | Laison-Brito, J. | De Castilho, P. V. | Daura-Jorge, F. G. | Simões-Lopes, P. C. | Carballo Santaolalla, Matilde | García-Parraga, Daniel | Sánchez-Vizcaíno, J. M. | Esperón, Fernando | Ewbank, Ana Carolina [0000-0002-5617-9287] | Ferreira-Machado, Eduardo [0000-0002-4610-7490] | García-Parraga, Daniel [0000-0002-3335-5831] | Neves, Elena [0000-0002-1814-573X] | Sacristán, Carlos [0000-0002-6111-6301] | Esperón, Fernando [0000-0002-8810-5071]
Poxviruses are emerging pathogens in cetaceans, temporarily named ‘Cetaceanpoxvirus’ (CePV, family Poxviridae), classified into two main lineages: CePV-1 in odontocetes and CePV-2 in mysticetes. Only a few studies performed the molecular detection of CePVs, based on DNA-polymerase gene and/or DNA-topoisomerase I gene amplification. Herein we describe a new real-time PCR assay based on SYBR® Green and a new primer set to detect a 150 bp fragment of CePV DNA-polymerase gene, also effective for conventional PCR detection. The novel real-time PCR was able to detect 5 up to 5×106 copies per reaction of a cloned positive control. Both novel PCR methods were 1000 to 100,000-fold more sensitive than those previously described in the literature. Samples of characteristic poxvirus skin lesions (‘tattoo’) from one Risso's dolphin (Grampus griseus), two striped dolphins (Stenella coeruleoalba) and two Guiana dolphins (Sotalia guianensis) were all positive to both our novel real time- and conventional PCR methods, even though three of these animals (a Risso's dolphin, a striped dolphin, and a Guiana dolphin) were previously negative to the conventional PCRs previously available. To our knowledge, this is the first real-time PCR detection method for Cetaceanpoxvirus, a much more sensitive tool for the detection of CePV-1 infections.
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