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Extraction-free protocol combining proteinase K and heat inactivation for detection of SARS-CoV-2 by RT-qPCR

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dc.contributor.author Genoud, Valeria
dc.contributor.author Stortz, Martin
dc.contributor.author Waisman, Ariel
dc.contributor.author Berardino, Bruno G.
dc.contributor.author Verneri, Paula
dc.contributor.author Dansey, Virginia
dc.contributor.author Salvatori, Melina
dc.contributor.author Remes Lenicov, Federico
dc.contributor.author Levi, Valeria
dc.date.accessioned 2021-03-15T14:38:51Z
dc.date.available 2021-03-15T14:38:51Z
dc.date.issued 2021-02-26
dc.identifier.citation Genoud V, Stortz M, Waisman A, Berardino BG, Verneri P, Dansey V, Salvatori M, Remes Lenicov F, Levi V. Extraction-free protocol combining proteinase K and heat inactivation for detection of SARS-CoV-2 by RT-qPCR. PLoS One. 2021 Feb 26;16(2):e0247792. doi: 10.1371/journal.pone.0247792 es_ES
dc.identifier.uri https://doi.org/10.1371/journal.pone.0247792
dc.identifier.uri https://repositorio.fleni.org.ar/xmlui/handle/123456789/377
dc.description.abstract Real-time reverse transcription PCR (RT-qPCR) is the gold-standard technique for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in nasopharyngeal swabs specimens. The analysis by RT-qPCR usually requires a previous extraction step to obtain the purified viral RNA. Unfortunately, RNA extraction constitutes a bottleneck for early detection in many countries since it is expensive, time-consuming and depends on the availability of commercial kits. Here, we describe an extraction-free protocol for SARS-CoV-2 detection by RT-qPCR from nasopharyngeal swab clinical samples in saline solution. The method includes a treatment with proteinase K followed by heat inactivation (PK+HID method). We demonstrate that PK+HID improves the RT-qPCR performance in comparison to the heat-inactivation procedure. Moreover, we show that this extraction-free protocol can be combined with a variety of multiplexing RT-qPCR kits. The method combined with a multiplexing detection kit targeting N and ORF1ab viral genes showed a sensitivity of 0.99 and a specificity of 0.99 from the analysis of 106 positive and 106 negative clinical samples. In conclusion, PK+HID is a robust, fast and inexpensive procedure for extraction-free RT-qPCR determinations of SARS-CoV-2. The National Administration of Drugs, Foods and Medical Devices of Argentina has recently authorized the use of this method. es_ES
dc.language.iso eng es_ES
dc.publisher PLoS One es_ES
dc.rights info:eu-repo/semantics/openAccess
dc.rights.uri https://creativecommons.org/licenses/by/2.5/ar/
dc.subject COVID-19 es_ES
dc.subject Endopeptidase K es_ES
dc.subject Endopeptidasa K es_ES
dc.subject Molecular Diagnostic Techniques es_ES
dc.subject Técnicas de Diagnóstico Molecular es_ES
dc.subject Nucleic Acid Amplification Techniques es_ES
dc.subject Técnicas de Amplificación de Ácido Nucleico es_ES
dc.subject SARS-CoV-2 es_ES
dc.title Extraction-free protocol combining proteinase K and heat inactivation for detection of SARS-CoV-2 by RT-qPCR es_ES
dc.type info:eu-repo/semantics/article es_ES
dc.type info:eu-repo/semantics/publishedVersion
dc.description.fil Fil: Genoud, Valeria. Universidad de Buenos Aire. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina.
dc.description.fil Fil: Stortz, Martin. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales, Departamento de Fisiología, Biología Molecular y Celular. Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales, Instituto de Química Biológica. Argentina.
dc.description.fil Fil: Waisman, Ariel. Fleni. Laboratorio de Investigación Aplicada a las Neurociencias; Argentina.
dc.description.fil Fil: Berardino, Bruno G. Universidad de Buenos Aire. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales, Instituto de Química Biológica. Argentina.
dc.description.fil Fil: Verneri, Paula. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales, Instituto de Química Biológica. Argentina.
dc.description.fil Fil: Dansey, Virginia. Universidad de Buenos Aire. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Argentina.
dc.description.fil Fil: Salvatori, Melina. Instituto de Investigaciones Biomédicas en Retrovirus y SIDA. Argentina.
dc.description.fil Fil: Remes Lenicov, Federico. Instituto de Investigaciones Biomédicas en Retrovirus y SIDA. Argentina.
dc.description.fil Fil: Levi, Valeria. Universidad de Buenos Aire. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales, Instituto de Química Biológica. Argentina.
dc.relation.ispartofVOLUME 16
dc.relation.ispartofNUMBER 2
dc.relation.ispartofPAGINATION e0247792
dc.relation.ispartofCOUNTRY Estados Unidos
dc.relation.ispartofCITY San Francisco
dc.relation.ispartofTITLE PLoS One
dc.relation.ispartofISSN 1932-6203
dc.type.snrd info:ar-repo/semantics/artículo es_ES


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