Novel and highly sensitive sybr? green real-time pcr for poxvirus detection in odontocete cetaceans
Investigation published in Journal of Virological Methods
September 1st, 2018
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
Sacristan C., Luiz Catao-Dias J., Ewbank AC., Machado EF., Neves E., Santos-Neto EB., Avezedo A., Laison-Brito J., De Castillo PV., Daura-Jorge FG., Simoes-Lopes PC., Carballo M., Garcia-Parraga D., Sanchez-Vizcaino JM. and Esperon F.
 | Laboratório de Patologia Comparada de Animais Selvagens. Faculdade de Medicina Veterinária e Zootecnia. Universidade de Sao Paulo (USP). |
 | Centro de Investigación en Sanidad Animal (CISA). Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA). |
| Laboratório de Mamíferos Aquáticos e Bioindicadores (MAQUA). Faculdade De Oceanografia. Universidade do Estado do Rio de Janeiro. | |
 | Departamento de Engenharia de Pesca. Universidade Federal de Santa Catarina (UFSC). |
| Departamento de Ecologia e Zoologia. Universidade Federal de Santa Catarina (UFSC). |
 | Veterinary Services. Oceanogràfic. Ciudad de las Artes y las Ciencias. Generalitat Valenciana. |
 | Servicio de Inmunología Viral y Medicina Preventiva (SUAT). Centro de Vigilancia Sanitaria Veterinaria (VISAVET). Universidad Complutense (UCM). |
| Departamento de Sanidad Animal. Facultad de Veterinaria. Universidad Complutense (UCM). |
