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A quadruplex qPCR for detection and differentiation of classic and natural recombinant Myxoma Virus Strains of leporids

2021-11-07Artigo científico Acesso aberto
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dc.contributor.authorAbade Dos Santos, F.A.
dc.contributor.authorCarvalho, Carina L.
dc.contributor.authorFrancisco, Parra
dc.contributor.authorDalton, Kevin P.
dc.contributor.authorPeleteiro, Maria C.
dc.contributor.authorDuarte, M. D.
dc.date.accessioned2022-01-07T12:00:37Z
dc.date.available2022-01-07T12:00:37Z
dc.date.issued2021-11-07
dc.descriptionResearch Areas: Biochemistry & Molecular Biology ; Chemistrypt_PT
dc.description.abstractA natural recombinant myxoma virus (referred to as ha-MYXV or MYXV-Tol08/18) emerged in the Iberian hare (Lepus granatensis) and the European rabbit (Oryctolagus cuniculus) 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 m009L gene that disrupted it into ORFs m009L-a and m009L-b. 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 m009L-a and m009L-b, only contiguous in classic strains, while the second amplifies a fragment within gene m060L, only present in recombinant MYXV strains. All amplification reactions were validated and normalized by a fourth PCR system directed to a housekeeping gene (18S rRNA) conserved in eukaryotic organisms, including hares and rabbits. The multiplex PCR (mPCR) technique described here was optimized for Taqman® and Evagreen® 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.pt_PT
dc.description.versioninfo:eu-repo/semantics/publishedVersionpt_PT
dc.identifier.citationAbade dos Santos FA, Carvalho CL, Parra F, Dalton KP, Peleteiro MC, Duarte, MD. 2021. A quadruplex qPCR for detection and differentiation of classic and natural recombinant Myxoma Virus Strains of leporids. International Journal of Molecular Sciences. 22(21):12052. DOI: 10.3390/ijms222112052pt_PT
dc.identifier.doi10.3390/ijms222112052pt_PT
dc.identifier.eissn1422-0067
dc.identifier.urihttp://hdl.handle.net/10400.5/22938
dc.language.isoengpt_PT
dc.peerreviewedyespt_PT
dc.publisherMDPIpt_PT
dc.relationUIDB/CVT/00276/2020pt_PT
dc.relationRabbit Viral Haemorrhagic Disease control in Iberian ecosystems: dynamics of virus spread, pathophysiology and improvement of prophylactic measures.
dc.relationAGL2017-83395-Rpt_PT
dc.relationPID2020-120349RB-100pt_PT
dc.relation.publisherversionhttps://www.mdpi.com/1422-0067/22/21/12052pt_PT
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/pt_PT
dc.subjectMolecular diagnosispt_PT
dc.subjectMultiplexpt_PT
dc.subjectQuadruplex qPCRpt_PT
dc.subjectReal-time PCRpt_PT
dc.subjectMyxoma viruspt_PT
dc.subjectMYXVpt_PT
dc.subjectNatural recombinant MYXVpt_PT
dc.subjectha-MYXVpt_PT
dc.subjectIberian harept_PT
dc.subjectEuropean rabbitpt_PT
dc.titleA quadruplex qPCR for detection and differentiation of classic and natural recombinant Myxoma Virus Strains of leporidspt_PT
dc.typejournal article
dspace.entity.typePublication
oaire.awardNumberSFRH/BD/137067/2018
oaire.awardTitleRabbit Viral Haemorrhagic Disease control in Iberian ecosystems: dynamics of virus spread, pathophysiology and improvement of prophylactic measures.
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/OE/SFRH%2FBD%2F137067%2F2018/PT
oaire.citation.conferencePlaceBasel, Switzerlandpt_PT
oaire.citation.titleInternational Journal of Molecular Sciencespt_PT
oaire.citation.volume22, 12052pt_PT
oaire.fundingStreamOE
person.familyNameAbade dos Santos
person.familyNamePeleteiro
person.familyNameDuarte
person.givenNameFábio
person.givenNameMaria
person.givenNameMargarida
person.identifier.ciencia-idD810-0FEC-8051
person.identifier.ciencia-id6317-1F85-5E59
person.identifier.orcid0000-0002-0696-7322
person.identifier.orcid0000-0003-1848-558X
person.identifier.orcid0000-0003-1488-9659
person.identifier.ridJ-2640-2013
person.identifier.scopus-author-id6602767105
person.identifier.scopus-author-id28267670300
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.nameFundação para a Ciência e a Tecnologia
rcaap.rightsopenAccesspt_PT
rcaap.typearticlept_PT
relation.isAuthorOfPublicationacec0d1a-7e06-43bb-9140-94ae67608b0f
relation.isAuthorOfPublication330bcdd7-5df6-4b33-94a4-b70d4ae6efd2
relation.isAuthorOfPublication28463e5f-2d1b-460b-b101-a409c4feffb4
relation.isAuthorOfPublication.latestForDiscoveryacec0d1a-7e06-43bb-9140-94ae67608b0f
relation.isProjectOfPublicatione4f33419-21df-431f-8c77-051faa708c0b
relation.isProjectOfPublication.latestForDiscoverye4f33419-21df-431f-8c77-051faa708c0b

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