A Quadruplex qPCR for Detection and Differentiation of Classic and Natural Recombinant Myxoma Virus Strains of Leporids
A natural recombinant myxoma virus (referred to as ha-MYXV or MYXV-Tol08/18) emerged in the Iberian hare (<i>Lepus granatensis</i>) and the European rabbit (<i>Oryctolagus cuniculus</i>) in late 2018 and mid-2020, respectively. This new virus is genetically distinct from clas...
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| Autores principales: | , , , , , |
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| Formato: | article |
| Lenguaje: | EN |
| Publicado: |
MDPI AG
2021
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| Materias: | |
| Acceso en línea: | https://doaj.org/article/3a6fc2953e924f85b4565e5c1589b0d0 |
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| Sumario: | A natural recombinant myxoma virus (referred to as ha-MYXV or MYXV-Tol08/18) emerged in the Iberian hare (<i>Lepus granatensis</i>) and the European rabbit (<i>Oryctolagus cuniculus</i>) in late 2018 and mid-2020, respectively. This new virus is genetically distinct from classic myxoma virus (MYXV) strains that caused myxomatosis in rabbits until then, by acquiring an additional 2.8 Kbp insert within the <i>m009L</i> gene that disrupted it into ORFs <i>m009L-a</i> and <i>m009L-b</i>. To distinguish ha-MYXV from classic MYXV strains, we developed a robust qPCR multiplex technique that combines the amplification of the m000.5L/R duplicated gene, conserved in all myxoma virus strains including ha-MYXV, with the amplification of two other genes targeted by the real-time PCR systems designed during this study, specific either for classic MYXV or ha-MYXV strains. The first system targets the boundaries between ORFs <i>m009L-a</i> and <i>m009L-b</i>, only contiguous in classic strains, while the second amplifies a fragment within gene <i>m060L</i>, only present in recombinant MYXV strains. All amplification reactions were validated and normalized by a fourth PCR system directed to a housekeeping gene (<i>18S rRNA</i>) conserved in eukaryotic organisms, including hares and rabbits. The multiplex PCR (mPCR) technique described here was optimized for <i>Taqman</i><sup>®</sup> and <i>Evagreen</i><sup>®</sup> systems allowing the detection of as few as nine copies of viral DNA in the sample with an efficiency > 93%. This real-time multiplex is the first fast method available for the differential diagnosis between classic and recombinant MYXV strains, also allowing the detection of co-infections. The system proves to be an essential and effective tool for monitoring the geographical spread of ha-MYXV in the hare and wild rabbit populations, supporting the management of both species in the field. |
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