Artículo
Extraction-free protocol combining proteinase K and heat inactivation for detection of SARS-CoV-2 by RT-qPCR
Genoud, Valeria; Stortz, Martin Dario
; Waisman, Ariel
; Berardino, Bruno Gabriel
; Verneri, Paula
; Dansey, Maria Virginia
; Salvatori, Melina
; Remes Lenicov, Federico
; Levi, Valeria
Fecha de publicación:
26/02/2021
Editorial:
Public Library of Science
Revista:
Plos One
ISSN:
1932-6203
Idioma:
Inglés
Tipo de recurso:
Artículo publicado
Clasificación temática:
Resumen
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.
Palabras clave:
SARS-COV-2
,
RNA EXTRACTION
,
PROTEASES
,
COVID-19
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Articulos(INBIRS)
Articulos de INSTITUTO DE INVESTIGACIONES BIOMEDICAS EN RETROVIRUS Y SIDA
Articulos de INSTITUTO DE INVESTIGACIONES BIOMEDICAS EN RETROVIRUS Y SIDA
Articulos(IQUIBICEN)
Articulos de INSTITUTO DE QUIMICA BIOLOGICA DE LA FACULTAD DE CS. EXACTAS Y NATURALES
Articulos de INSTITUTO DE QUIMICA BIOLOGICA DE LA FACULTAD DE CS. EXACTAS Y NATURALES
Articulos(UMYMFOR)
Articulos de UNID.MICROANAL.Y MET.FISICOS EN QUIM.ORG.(I)
Articulos de UNID.MICROANAL.Y MET.FISICOS EN QUIM.ORG.(I)
Citación
Genoud, Valeria; Stortz, Martin Dario; Waisman, Ariel; Berardino, Bruno Gabriel; Verneri, Paula; et al.; Extraction-free protocol combining proteinase K and heat inactivation for detection of SARS-CoV-2 by RT-qPCR; Public Library of Science; Plos One; 16; 2; 26-2-2021; 1-16
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