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Cell-based internal standard for qPCR determinations of antibiotic resistance indicators in environmental water samples

2020Artigo científico Acesso aberto
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dc.contributor.authorRocha, Jaqueline
dc.contributor.authorManaia, Célia M.
dc.date.accessioned2020-03-10T18:34:16Z
dc.date.available2020-03-10T18:34:16Z
dc.date.issued2020
dc.description.abstractQuantitative PCR (qPCR) has been used to quantify antibiotic resistance genes (ARGs) in water, wastewater, soil, sediment and tissue samples. Concerns regarding the comparability of data obtained in different laboratories has been a major bottleneck to incentivize the compilation of publicly available of ARGs quantifications gathered from different reports. In this study, the influence of the DNA extraction kits (NZY Tissue gDNA Isolation kit or DNeasy PowerWater kit) and of the operator on the DNA extraction yield and on qPCR genes quantification was assessed. Since in wastewater and water samples the matrix effect can affect the DNA recovery and, therefore, gene quantification, an internal standard, consisting in a cloned gene not found in environmental samples, was tested. The aim was to assess how qPCR determinations in wastewater and water samples can be affected by the matrix effect. The results show that the DNA extraction operator did not significantly influence DNA yield. The use of distinct kits resulted in qPCR gene quantifications that did not differ in more than 1 log-unit mL−1. The matrix effect, assessed based on the use of an internal standard, was associated with an underestimation that ranged 0.1–0.9 log gene copy number mL−1 of sample, irrespective of the water type. The reliability on the use of a DNA extraction kit that costs about 3 times less than the most commonly used can be an incentive for the use of DNA based analyses of ARGs in environmental waters. Moreover, the fact that both the DNA extraction operator and the reduced matrix effect have little influence on the final results, are good news, encouraging the compilation of data produced in distinct laboratories. Nevertheless, harmonization efforts are still necessary to minimize bias that may be due associated with other conditions, such as equipment.pt_PT
dc.description.versioninfo:eu-repo/semantics/publishedVersionpt_PT
dc.identifier.citationRocha, J., Manaia, C. M. (2020). Cell-based internal standard for qPCR determinations of antibiotic resistance indicators in environmental water samples. Ecological Indicators, 113: 106194pt_PT
dc.identifier.doi10.1016/j.ecolind.2020.106194pt_PT
dc.identifier.eid85079113386
dc.identifier.issn1470-160X
dc.identifier.urihttp://hdl.handle.net/10400.14/29900
dc.identifier.wos000523335900022
dc.language.isoengpt_PT
dc.peerreviewedyespt_PT
dc.publisherElsevierpt_PT
dc.relationNORTE-01-0247-FEDER-033330pt_PT
dc.relationCentre for Biotechnology and Fine Chemistry
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/4.0/
dc.subjectQuantitative PCRpt_PT
dc.subjectDNA extractionpt_PT
dc.subjectInternal standardpt_PT
dc.subjectMatrix effectpt_PT
dc.titleCell-based internal standard for qPCR determinations of antibiotic resistance indicators in environmental water samplespt_PT
dc.typejournal article
dspace.entity.typePublication
oaire.awardNumberUID/Multi/50016/2019
oaire.awardNumberWaterJPI/0001/2013
oaire.awardTitleCentre for Biotechnology and Fine Chemistry
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UID%2FMulti%2F50016%2F2019/PT
oaire.awardURIinfo:eu-repo/grantAgreement/FCT/3599-PPCDT/WaterJPI%2F0001%2F2013/PT
oaire.citation.titleEcological Indicatorspt_PT
oaire.citation.volume113pt_PT
oaire.fundingStream6817 - DCRRNI ID
oaire.fundingStream3599-PPCDT
person.familyNameRocha
person.familyNameManaia
person.givenNameJaqueline
person.givenNameCélia
person.identifier.ciencia-id721C-C3E4-6A38
person.identifier.ciencia-idC31F-553B-6365
person.identifier.orcid0000-0003-1651-9483
person.identifier.orcid0000-0002-3674-1789
person.identifier.ridK-3850-2014
person.identifier.scopus-author-id57113035100
person.identifier.scopus-author-id6602465318
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.identifierhttp://doi.org/10.13039/501100001871
project.funder.nameFundação para a Ciência e a Tecnologia
project.funder.nameFundação para a Ciência e a Tecnologia
rcaap.rightsopenAccesspt_PT
rcaap.typearticlept_PT
relation.isAuthorOfPublication61cb19ec-ea8b-4294-a77d-378aa7994f16
relation.isAuthorOfPublicationa5f14577-0aed-4046-97da-5f4216c7aa07
relation.isAuthorOfPublication.latestForDiscoverya5f14577-0aed-4046-97da-5f4216c7aa07
relation.isProjectOfPublication1f76a7db-2520-482f-b56b-7e01ea36dd1f
relation.isProjectOfPublication8e6c9eb2-1cfa-4686-bf25-86aacc2217fb
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