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Development of a quantitative one-step multiplex RT-qPCR assay for the detection of SARS-CoV-2 in a biological matrix

INTRODUCTION: Coronavirus disease-2019 (COVID-19) is a disease caused by Severe Acute Respiratory Syndrome Virus 2 (SARS-CoV-2) that emerged in China in late 2019. The rapid viral spread has made the disease a public health emergency of worldwide concern. The gold standard for diagnosing SARS-CoV-2...

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Autores principales: Queiroz, Jackson Alves da Silva, Rampazzo, Rita de Cássia Pontello, Filho, Edivá Basílio da Silva, Oliveira, Gabriella Sgorlon, Oliveira, Suyane da Costa, Souza, Luan Felipo Botelho, Pereira, Soraya dos Santos, Rodrigues, Moreno Magalhães de Souza, Maia, Adriana Cristina Salvador, da Silva, Cicileia Correia, Mendonça, Aline Linhares Ferreira de Melo, Lugtenburg, Celina Aparecida Bertoni, Aguiar, Francisco de Assis Araújo, Rodrigues, Rosiane de Souza Soares, Santos, Caio Henrique Nemeth, Guimarães, Alice Paula Di Sabatino, Máximo, Fernando Rodrigues, Santos, Alcione de Oliveira dos, Krieger, Marco Aurélio, Salcedo, Juan Miguel Villalobos, Dall’Acqua, Deusilene Souza Vieira
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7831874/
https://www.ncbi.nlm.nih.gov/pubmed/33434663
http://dx.doi.org/10.1016/j.ijid.2021.01.001
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author Queiroz, Jackson Alves da Silva
Rampazzo, Rita de Cássia Pontello
Filho, Edivá Basílio da Silva
Oliveira, Gabriella Sgorlon
Oliveira, Suyane da Costa
Souza, Luan Felipo Botelho
Pereira, Soraya dos Santos
Rodrigues, Moreno Magalhães de Souza
Maia, Adriana Cristina Salvador
da Silva, Cicileia Correia
Mendonça, Aline Linhares Ferreira de Melo
Lugtenburg, Celina Aparecida Bertoni
Aguiar, Francisco de Assis Araújo
Rodrigues, Rosiane de Souza Soares
Santos, Caio Henrique Nemeth
Guimarães, Alice Paula Di Sabatino
Máximo, Fernando Rodrigues
Santos, Alcione de Oliveira dos
Krieger, Marco Aurélio
Salcedo, Juan Miguel Villalobos
Dall’Acqua, Deusilene Souza Vieira
author_facet Queiroz, Jackson Alves da Silva
Rampazzo, Rita de Cássia Pontello
Filho, Edivá Basílio da Silva
Oliveira, Gabriella Sgorlon
Oliveira, Suyane da Costa
Souza, Luan Felipo Botelho
Pereira, Soraya dos Santos
Rodrigues, Moreno Magalhães de Souza
Maia, Adriana Cristina Salvador
da Silva, Cicileia Correia
Mendonça, Aline Linhares Ferreira de Melo
Lugtenburg, Celina Aparecida Bertoni
Aguiar, Francisco de Assis Araújo
Rodrigues, Rosiane de Souza Soares
Santos, Caio Henrique Nemeth
Guimarães, Alice Paula Di Sabatino
Máximo, Fernando Rodrigues
Santos, Alcione de Oliveira dos
Krieger, Marco Aurélio
Salcedo, Juan Miguel Villalobos
Dall’Acqua, Deusilene Souza Vieira
author_sort Queiroz, Jackson Alves da Silva
collection PubMed
description INTRODUCTION: Coronavirus disease-2019 (COVID-19) is a disease caused by Severe Acute Respiratory Syndrome Virus 2 (SARS-CoV-2) that emerged in China in late 2019. The rapid viral spread has made the disease a public health emergency of worldwide concern. The gold standard for diagnosing SARS-CoV-2 is reverse transcription followed by qualitative real-time polymerase chain reaction (RT-qPCR); however, the role of viral load quantification has not been thoroughly investigated yet. OBJECTIVE: The aim of this study was to develop a high-precision quantitative one-step RT-qPCR reaction using the association of the viral target and the human target in the same reaction. METHODS: The assay standardization involved the absolute quantification method, with serial dilutions of a plasmid with the N gene in a biological matrix to build a standard curve. RESULTS AND DISCUSSION: The results demonstrated the possibility of quantifying as few as 2.5 copies/reaction and an analysis of 244 patients with known results selected by cross-section that revealed 100% agreement with a qualitative RT-qPCR assay registered by Anvisa. In this population, it was possible to quantify patients with between 2.59 and 3.5 × 10(7) copies per reaction and negative patients continued to indicate the same result. CONCLUSION: This assay can be a useful tool for a proper patient management, because the level and duration of viral replication are important factors to assess the risk of transmission and to guide decisions regarding the isolation and release of patients; an accurate diagnosis is critical information, whereas the current COVID-19 pandemic represents the biggest current global health problem.
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spelling pubmed-78318742021-01-26 Development of a quantitative one-step multiplex RT-qPCR assay for the detection of SARS-CoV-2 in a biological matrix Queiroz, Jackson Alves da Silva Rampazzo, Rita de Cássia Pontello Filho, Edivá Basílio da Silva Oliveira, Gabriella Sgorlon Oliveira, Suyane da Costa Souza, Luan Felipo Botelho Pereira, Soraya dos Santos Rodrigues, Moreno Magalhães de Souza Maia, Adriana Cristina Salvador da Silva, Cicileia Correia Mendonça, Aline Linhares Ferreira de Melo Lugtenburg, Celina Aparecida Bertoni Aguiar, Francisco de Assis Araújo Rodrigues, Rosiane de Souza Soares Santos, Caio Henrique Nemeth Guimarães, Alice Paula Di Sabatino Máximo, Fernando Rodrigues Santos, Alcione de Oliveira dos Krieger, Marco Aurélio Salcedo, Juan Miguel Villalobos Dall’Acqua, Deusilene Souza Vieira Int J Infect Dis Article INTRODUCTION: Coronavirus disease-2019 (COVID-19) is a disease caused by Severe Acute Respiratory Syndrome Virus 2 (SARS-CoV-2) that emerged in China in late 2019. The rapid viral spread has made the disease a public health emergency of worldwide concern. The gold standard for diagnosing SARS-CoV-2 is reverse transcription followed by qualitative real-time polymerase chain reaction (RT-qPCR); however, the role of viral load quantification has not been thoroughly investigated yet. OBJECTIVE: The aim of this study was to develop a high-precision quantitative one-step RT-qPCR reaction using the association of the viral target and the human target in the same reaction. METHODS: The assay standardization involved the absolute quantification method, with serial dilutions of a plasmid with the N gene in a biological matrix to build a standard curve. RESULTS AND DISCUSSION: The results demonstrated the possibility of quantifying as few as 2.5 copies/reaction and an analysis of 244 patients with known results selected by cross-section that revealed 100% agreement with a qualitative RT-qPCR assay registered by Anvisa. In this population, it was possible to quantify patients with between 2.59 and 3.5 × 10(7) copies per reaction and negative patients continued to indicate the same result. CONCLUSION: This assay can be a useful tool for a proper patient management, because the level and duration of viral replication are important factors to assess the risk of transmission and to guide decisions regarding the isolation and release of patients; an accurate diagnosis is critical information, whereas the current COVID-19 pandemic represents the biggest current global health problem. The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. 2021-03 2021-01-09 /pmc/articles/PMC7831874/ /pubmed/33434663 http://dx.doi.org/10.1016/j.ijid.2021.01.001 Text en © 2021 The Authors Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.
spellingShingle Article
Queiroz, Jackson Alves da Silva
Rampazzo, Rita de Cássia Pontello
Filho, Edivá Basílio da Silva
Oliveira, Gabriella Sgorlon
Oliveira, Suyane da Costa
Souza, Luan Felipo Botelho
Pereira, Soraya dos Santos
Rodrigues, Moreno Magalhães de Souza
Maia, Adriana Cristina Salvador
da Silva, Cicileia Correia
Mendonça, Aline Linhares Ferreira de Melo
Lugtenburg, Celina Aparecida Bertoni
Aguiar, Francisco de Assis Araújo
Rodrigues, Rosiane de Souza Soares
Santos, Caio Henrique Nemeth
Guimarães, Alice Paula Di Sabatino
Máximo, Fernando Rodrigues
Santos, Alcione de Oliveira dos
Krieger, Marco Aurélio
Salcedo, Juan Miguel Villalobos
Dall’Acqua, Deusilene Souza Vieira
Development of a quantitative one-step multiplex RT-qPCR assay for the detection of SARS-CoV-2 in a biological matrix
title Development of a quantitative one-step multiplex RT-qPCR assay for the detection of SARS-CoV-2 in a biological matrix
title_full Development of a quantitative one-step multiplex RT-qPCR assay for the detection of SARS-CoV-2 in a biological matrix
title_fullStr Development of a quantitative one-step multiplex RT-qPCR assay for the detection of SARS-CoV-2 in a biological matrix
title_full_unstemmed Development of a quantitative one-step multiplex RT-qPCR assay for the detection of SARS-CoV-2 in a biological matrix
title_short Development of a quantitative one-step multiplex RT-qPCR assay for the detection of SARS-CoV-2 in a biological matrix
title_sort development of a quantitative one-step multiplex rt-qpcr assay for the detection of sars-cov-2 in a biological matrix
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7831874/
https://www.ncbi.nlm.nih.gov/pubmed/33434663
http://dx.doi.org/10.1016/j.ijid.2021.01.001
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