A novel HRM assay for the simultaneous detection and differentiation of eight poxviruses of medical and veterinary importance

Esayas Gelaye, Lukas Mach, Jolanta Kolodziejek, Reingard Grabherr, Angelika Loitsch, Jenna E. Achenbach, Norbert Nowotny, Adama Diallo, Charles Euloge Lamien

Research output: Contribution to journalArticle

9 Citations (Scopus)

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.

Original languageEnglish
Article number42892
JournalScientific Reports
Volume7
DOIs
Publication statusPublished - Feb 20 2017

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Pseudocowpox virus
Camelpox virus
Poxviridae
Bovine papular stomatitis virus
Goatpox virus
Lumpy skin disease virus
Capripoxvirus
Sheeppox virus
Cowpox virus
Parapoxvirus
Orf virus
Orthopoxvirus
melting
assays
dye binding
melting point
mixed infection
genotyping
sampling
DNA

ASJC Scopus subject areas

  • General

Cite this

Gelaye, E., Mach, L., Kolodziejek, J., Grabherr, R., Loitsch, A., Achenbach, J. E., ... Lamien, C. E. (2017). A novel HRM assay for the simultaneous detection and differentiation of eight poxviruses of medical and veterinary importance. Scientific Reports, 7, [42892]. https://doi.org/10.1038/srep42892

A novel HRM assay for the simultaneous detection and differentiation of eight poxviruses of medical and veterinary importance. / Gelaye, Esayas; Mach, Lukas; Kolodziejek, Jolanta; Grabherr, Reingard; Loitsch, Angelika; Achenbach, Jenna E.; Nowotny, Norbert; Diallo, Adama; Lamien, Charles Euloge.

In: Scientific Reports, Vol. 7, 42892, 20.02.2017.

Research output: Contribution to journalArticle

Gelaye, E, Mach, L, Kolodziejek, J, Grabherr, R, Loitsch, A, Achenbach, JE, Nowotny, N, Diallo, A & Lamien, CE 2017, 'A novel HRM assay for the simultaneous detection and differentiation of eight poxviruses of medical and veterinary importance', Scientific Reports, vol. 7, 42892. https://doi.org/10.1038/srep42892
Gelaye, Esayas ; Mach, Lukas ; Kolodziejek, Jolanta ; Grabherr, Reingard ; Loitsch, Angelika ; Achenbach, Jenna E. ; Nowotny, Norbert ; Diallo, Adama ; Lamien, Charles Euloge. / A novel HRM assay for the simultaneous detection and differentiation of eight poxviruses of medical and veterinary importance. In: Scientific Reports. 2017 ; Vol. 7.
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