Novel and highly sensitive SYBR® Green real-time pcr for poxvirus detection in odontocete cetaceans

Abstract

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 × 10 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.

The Pathology of Cetacean Morbillivirus Infection and Comorbidities in Guiana Dolphins During an Unusual Mortality Event (Brazil, 2017–2018)

“…Herpesviral detection followed the protocol and primers described by VanDevanter et al 71 for the DNA polymerase gene and by Ehlers et al 25 for the glycoprotein B gene with slight modifications. Two different conventional PCR protocols to partially amplify the cetaceanpoxvirus DNA polymerase gene 8,53 were employed to detect poxvirus. Herpesvirus-PCR-positive controls included a commercial feline vaccine against feline herpesvirus 1 (Nobivac Feline1-HCPCh, MSD) for the DNA polymerase gene and a skin sample from a herpesvirus-positive Guiana dolphin for the glycoprotein B gene.…”

The Validation of a Non-Invasive Skin Sampling Device for Detecting Cetacean Poxvirus

“…Hence, the same skin sampling procedure using CCS explained above was performed repeatedly. The CePV-1 positive control was a 0.025 g biopsy that had previously been confirmed by our group using the real-time PCR method [63] and sequencing amplicons (unpublished sequencing results) previously described. Once 1 mL RNAlater-epidermis mixtures were obtained, they were subsequently subjected to a high centrifugation speed for 5 min (14,000 rpm).…”

“…The molecular detection of CePV-1 was performed using a 1-step real-time polymerase chain (q-PCR) method to amplify a conserved region (150 bp) of the DNA polymerase gene by using the degenerate primer sets designed by Sacristán et al [63] (Odontopox-F: 5 -CARGAAATMAAAAAGAARTTTCCATC-3 , and Odontopox-R: 5 -ACGTTCTGTTAARA AYCGTCTTAGTA-3 ). The thermocycler profile was set for initial denaturation at 95 • C for 5 min, followed by 40 amplification cycles, each compromised of a denaturation step at 95 • C for 15 s, an annealing step at 60 • C for 30 s, and an elongation step at 72 • C for 30 s. The final cycle was composed of an extended elongation, which was performed at 72 • C for 7 min [29].…”

Towards understanding host–pathogen dynamics of cetacean poxvirus: attainable approach through the application of a repetitive non-invasive skin sampling in bottlenose dolphins (Tursiops truncatus) under human care

“…DNA extraction from sloughed skin samples was carried out through the DNeasy ™ Blood and Tissue Kit (Qiagen, Inc., Valencia, CA, USA) with some adaptations as thoroughly explained in Segura-Göthlin et al (2021) (Segura-Göthlin et al, 2021). Subsequently, the molecular detection of CePV-1 was performed using a real-time polymerase chain reaction (q-PCR) method to amplify a conserved region (150 bp) of the DNA polymerase gene by using the degenerate primer sets designed by Sacristań et al (2018a) (Odontopox-F: 5'-CARGAAATMAAAAAGAARTTTCCATC-3', and Odontopox-R: 5'-ACGTTCTGTTAARA AYCGTCTTAGTA-3'). Negative (nucleasefree water) and positive controls previously confirmed by our group for both extraction and amplification were included.…”