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:	Fábio A. Abade dos Santos, Carina L. Carvalho, Francisco Parra, Kevin P. Dalton, Maria C. Peleteiro, Margarida D. Duarte
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	molecular diagnosis multiplex quadruplex qPCR real-time PCR <i>myxoma virus</i> MYXV Biology (General) QH301-705.5 Chemistry QD1-999
Acceso en línea:	https://doaj.org/article/3a6fc2953e924f85b4565e5c1589b0d0
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spelling	oai:doaj.org-article:3a6fc2953e924f85b4565e5c1589b0d02021-11-11T17:26:52ZA Quadruplex qPCR for Detection and Differentiation of Classic and Natural Recombinant Myxoma Virus Strains of Leporids10.3390/ijms2221120521422-00671661-6596https://doaj.org/article/3a6fc2953e924f85b4565e5c1589b0d02021-11-01T00:00:00Zhttps://www.mdpi.com/1422-0067/22/21/12052https://doaj.org/toc/1661-6596https://doaj.org/toc/1422-0067A 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.Fábio A. Abade dos SantosCarina L. CarvalhoFrancisco ParraKevin P. DaltonMaria C. PeleteiroMargarida D. DuarteMDPI AGarticlemolecular diagnosismultiplexquadruplex qPCRreal-time PCR<i>myxoma virus</i>MYXVBiology (General)QH301-705.5ChemistryQD1-999ENInternational Journal of Molecular Sciences, Vol 22, Iss 12052, p 12052 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	molecular diagnosis multiplex quadruplex qPCR real-time PCR <i>myxoma virus</i> MYXV Biology (General) QH301-705.5 Chemistry QD1-999
spellingShingle	molecular diagnosis multiplex quadruplex qPCR real-time PCR <i>myxoma virus</i> MYXV Biology (General) QH301-705.5 Chemistry QD1-999 Fábio A. Abade dos Santos Carina L. Carvalho Francisco Parra Kevin P. Dalton Maria C. Peleteiro Margarida D. Duarte A Quadruplex qPCR for Detection and Differentiation of Classic and Natural Recombinant Myxoma Virus Strains of Leporids
description	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.
format	article
author	Fábio A. Abade dos Santos Carina L. Carvalho Francisco Parra Kevin P. Dalton Maria C. Peleteiro Margarida D. Duarte
author_facet	Fábio A. Abade dos Santos Carina L. Carvalho Francisco Parra Kevin P. Dalton Maria C. Peleteiro Margarida D. Duarte
author_sort	Fábio A. Abade dos Santos
title	A Quadruplex qPCR for Detection and Differentiation of Classic and Natural Recombinant Myxoma Virus Strains of Leporids
title_short	A Quadruplex qPCR for Detection and Differentiation of Classic and Natural Recombinant Myxoma Virus Strains of Leporids
title_full	A Quadruplex qPCR for Detection and Differentiation of Classic and Natural Recombinant Myxoma Virus Strains of Leporids
title_fullStr	A Quadruplex qPCR for Detection and Differentiation of Classic and Natural Recombinant Myxoma Virus Strains of Leporids
title_full_unstemmed	A Quadruplex qPCR for Detection and Differentiation of Classic and Natural Recombinant Myxoma Virus Strains of Leporids
title_sort	quadruplex qpcr for detection and differentiation of classic and natural recombinant myxoma virus strains of leporids
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/3a6fc2953e924f85b4565e5c1589b0d0
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A Quadruplex qPCR for Detection and Differentiation of Classic and Natural Recombinant Myxoma Virus Strains of Leporids

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