Analytical and Clinical Validation for RT-qPCR Detection of SARS-CoV-2 Without RNA Extraction

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dc.contributor.authorMiranda Marín, José Patricio
dc.contributor.authorHenríquez Henríquez, Marcela Patricia
dc.contributor.authorOsorio, Javiera
dc.contributor.authorVidela, Mauricio
dc.contributor.authorÁngel, Gladys
dc.contributor.authorCamponovo, Rossana
dc.date.accessioned2020-11-17T13:43:05Z
dc.date.available2020-11-17T13:43:05Z
dc.date.issued2020
dc.date.updated2020-11-14T02:13:53Z
dc.description.abstractBackground: The recent COVID-19 pandemic has posed an unprecedented challenge to laboratory diagnosis, based on the amplification of SARS-CoV-2 RNA. With global contagion figures exceeding 4 million persons, the shortage of reagents for RNA extraction represents a bottleneck for testing globally. We present the validation results for an RT-qPCR protocol without prior RNA extraction. Due to its simplicity, this protocol is suitable for widespread application in resource-limited settings. Methods: Optimal direct protocol was selected by comparing RT-qPCR performance under a set of thermal (65, 70, and 95° for 5, 10, and 30 min) and amplification conditions (3 or 3.5 uL loading volume; 2 commercial RT-qPCR kits with a limit of detection below 10 copies/reaction) in nasopharyngeal swabs stored at 4°C in sterile Weise's buffer pH 7.2. The selected protocol was evaluated for classification concordance with a standard protocol (automated RNA extraction) in 130 routine samples and 50 historical samples with Cq values near to the clinical decision limit. Results: Optimal selected conditions for direct protocol were: thermal shock at 70°C for 10 min, loading 3.5 ul in the RT-qPCR. Prospective evaluation in 130 routine samples showed a 100% classification concordance with the standard protocol. The evaluation in historical samples, selected because their Cqs were at the clinical decision limit, showed 94% concordance with our confirmatory standard, which includes manual RNA extraction. Conclusions: Our results validate the use of this direct RT-qPCR protocol as a safe alternative for SARS-CoV-2 diagnosis in the case of a shortage of reagents for RNA extraction, with minimal clinical impact.
dc.format.extent9 páginas
dc.identifier.doi10.3389/fmed.2020.567572
dc.identifier.urihttps://doi.org/10.3389/fmed.2020.567572
dc.identifier.urihttps://repositorio.uc.cl/handle/11534/48347
dc.language.isoen
dc.pagina.final9
dc.pagina.inicio1
dc.revistaFrontiers in Medicinees_ES
dc.rightsacceso abierto
dc.subjectSARS-CoV-2es_ES
dc.subjectCOVID-19es_ES
dc.subjectDiagnostices_ES
dc.subjectDirect RT-qPCRes_ES
dc.subjectRNA extractiones_ES
dc.subjectPandemic (COVID-19)es_ES
dc.subject.ddc616.2
dc.subject.deweyMedicina y saludes_ES
dc.titleAnalytical and Clinical Validation for RT-qPCR Detection of SARS-CoV-2 Without RNA Extractiones_ES
dc.typeartículo
dc.volumenVol. 7
sipa.codpersvinculados1126717
sipa.codpersvinculados12299
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