title: A novel HRM assay for the simultaneous detection and differentiation of eight poxviruses of medical and veterinary importance.
abstract: Poxviruses belonging to the Orthopoxvirus, Capripoxvirus and Parapoxvirus genera share common host species and create a challenge for diagnosis. Here, we developed a novel multiplex PCR method for the simultaneous detection and differentiation of eight poxviruses, belonging to three genera: cowpox virus (CPXV) and camelpox virus (CMLV) [genus Orthopoxvirus]; goatpox virus (GTPV), sheeppox virus (SPPV) and lumpy skin disease virus (LSDV) [genus Capripoxvirus]; orf virus (ORFV), pseudocowpox virus (PCPV) and bovine papular stomatitis virus (BPSV) [genus Parapoxvirus]. The assay is based on high-resolution melting curve analysis (HRMCA) of PCR amplicons produced using genus specific primer pairs and dsDNA binding dye. Differences in fragment size and GC content were used as discriminating power. The assay generated three well separated melting regions for each genus and provided additional intra-genus genotyping allowing the differentiation of the eight poxviruses based on amplicon melting temperature. Out of 271 poxviral DNA samples tested: seven CPXV, 25 CMLV, 42 GTPV, 20 SPPV, 120 LSDV, 33 ORFV, 20 PCPV and two BPSV were detected; two samples presented co-infection with CMLV and PCPV. The assay provides a rapid, sensitive, specific and cost-effective method for the detection of pox diseases in a broad range of animal species and humans.
authors.affiliation: Animal Production and Health Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Wagramer Strasse 5, P.O. Box 100, A-1400 Vienna, Austria.|Institute of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria.|Research and Development Department, National Veterinary Institute, P.O. Box 19, Debre Zeit, Ethiopia.
authors.forename: Esayas
authors.initials: E
authors.lastname: Gelaye
authors.affiliation: Institute of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria.
authors.forename: Lukas
authors.initials: L
authors.lastname: Mach
authors.affiliation: Viral Zoonoses, Emerging and Vector-Borne Infections Group, Institute of Virology, University of Veterinary Medicine, Veterinaerplatz 1, A-1210 Vienna, Austria.
authors.forename: Jolanta
authors.initials: J
authors.lastname: Kolodziejek
authors.affiliation: Institute of Applied Microbiology, University of Natural Resources and Life Sciences, Muthgasse 11, A-1190 Vienna, Austria.
authors.forename: Reingard
authors.initials: R
authors.lastname: Grabherr
authors.affiliation: Institute for Veterinary Disease Control, Austrian Agency for Health and Food Safety, Robert Koch-Gasse 17, A-2340 Mödling, Austria.
authors.forename: Angelika
authors.initials: A
authors.lastname: Loitsch
authors.affiliation: Animal Production and Health Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Wagramer Strasse 5, P.O. Box 100, A-1400 Vienna, Austria.
authors.forename: Jenna E
authors.initials: JE
authors.lastname: Achenbach
authors.affiliation: Viral Zoonoses, Emerging and Vector-Borne Infections Group, Institute of Virology, University of Veterinary Medicine, Veterinaerplatz 1, A-1210 Vienna, Austria.|Department of Basic Medical Sciences, College of Medicine, Mohammed Bin Rashid University of Medicine and Health Sciences, Dubai Healthcare City, P.O. Box 505055, Dubai, United Arab Emirates.
authors.forename: Norbert
authors.initials: N
authors.lastname: Nowotny
authors.affiliation: Animal Production and Health Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Wagramer Strasse 5, P.O. Box 100, A-1400 Vienna, Austria.
authors.forename: Adama
authors.initials: A
authors.lastname: Diallo
authors.affiliation: Animal Production and Health Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Wagramer Strasse 5, P.O. Box 100, A-1400 Vienna, Austria.
authors.forename: Charles Euloge
authors.initials: CE
authors.lastname: Lamien
mesh_terms: D000818:Animals; D001482:Base Composition; D004279:DNA, Viral / Q000032:analysis; D005838:Genotype; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods* / Q000662:veterinary; D011212:Poxviridae / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D011213:Poxviridae Infections / Q000175:diagnosis* / Q000662:veterinary; D012680:Sensitivity and Specificity; D013045:Species Specificity; D044366:Transition Temperature
chemical_list: D004279:DNA, Viral