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Validation of conventional PCR-like alternative to SARS-CoV-2 detection with target nucleocapsid protein gene in naso-oropharyngeal samples

SARS-CoV-2 has spread worldwide and has become a global health problem. As a result, the demand for inputs for diagnostic tests rose dramatically, as did the cost. Countries with inadequate infrastructure experience difficulties in expanding their qPCR testing capacity. Therefore, the development of...

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Autores principales: Carvalho, Rogério Fernandes, Oliveira, Monike da Silva, Ribeiro, Juliane, dos Santos, Isac Gabriel Cunha, Almeida, Katyane de Sousa, Conti, Ana Carolina Muller, Alexandrino, Bruna, Campos, Fabrício Souza, Soares, Célia Maria de Almeida, Ribeiro Júnior, José Carlos
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8459981/
https://www.ncbi.nlm.nih.gov/pubmed/34555073
http://dx.doi.org/10.1371/journal.pone.0257350
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author Carvalho, Rogério Fernandes
Oliveira, Monike da Silva
Ribeiro, Juliane
dos Santos, Isac Gabriel Cunha
Almeida, Katyane de Sousa
Conti, Ana Carolina Muller
Alexandrino, Bruna
Campos, Fabrício Souza
Soares, Célia Maria de Almeida
Ribeiro Júnior, José Carlos
author_facet Carvalho, Rogério Fernandes
Oliveira, Monike da Silva
Ribeiro, Juliane
dos Santos, Isac Gabriel Cunha
Almeida, Katyane de Sousa
Conti, Ana Carolina Muller
Alexandrino, Bruna
Campos, Fabrício Souza
Soares, Célia Maria de Almeida
Ribeiro Júnior, José Carlos
author_sort Carvalho, Rogério Fernandes
collection PubMed
description SARS-CoV-2 has spread worldwide and has become a global health problem. As a result, the demand for inputs for diagnostic tests rose dramatically, as did the cost. Countries with inadequate infrastructure experience difficulties in expanding their qPCR testing capacity. Therefore, the development of sensitive and specific alternative methods is essential. This study aimed to develop, standardize, optimize, and validate conventional RT-PCR targeting the N gene of SARS-CoV-2 in naso-oropharyngeal swab samples compared to qPCR. Using bioinformatics tools, specific primers were determined, with a product expected to be 519 bp. The reaction conditions were optimized using a commercial positive control, and the detection limit was determined to be 100 fragments. To validate conventional RT-PCR, we determined a representative sampling of 346 samples from patients with suspected infection whose diagnosis was made in parallel with qPCR. A sensitivity of 92.1% and specificity of 100% were verified, with an accuracy of 95.66% and correlation coefficient of 0.913. Under current Brazilian conditions, this method generates approximately 60% savings compared to qPCR costs. Conventional RT-PCR, validated herein, showed sufficient results for the detection of SARS-CoV-2 and can be used as an alternative for epidemiological studies and interspecies correlations.
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spelling pubmed-84599812021-09-24 Validation of conventional PCR-like alternative to SARS-CoV-2 detection with target nucleocapsid protein gene in naso-oropharyngeal samples Carvalho, Rogério Fernandes Oliveira, Monike da Silva Ribeiro, Juliane dos Santos, Isac Gabriel Cunha Almeida, Katyane de Sousa Conti, Ana Carolina Muller Alexandrino, Bruna Campos, Fabrício Souza Soares, Célia Maria de Almeida Ribeiro Júnior, José Carlos PLoS One Research Article SARS-CoV-2 has spread worldwide and has become a global health problem. As a result, the demand for inputs for diagnostic tests rose dramatically, as did the cost. Countries with inadequate infrastructure experience difficulties in expanding their qPCR testing capacity. Therefore, the development of sensitive and specific alternative methods is essential. This study aimed to develop, standardize, optimize, and validate conventional RT-PCR targeting the N gene of SARS-CoV-2 in naso-oropharyngeal swab samples compared to qPCR. Using bioinformatics tools, specific primers were determined, with a product expected to be 519 bp. The reaction conditions were optimized using a commercial positive control, and the detection limit was determined to be 100 fragments. To validate conventional RT-PCR, we determined a representative sampling of 346 samples from patients with suspected infection whose diagnosis was made in parallel with qPCR. A sensitivity of 92.1% and specificity of 100% were verified, with an accuracy of 95.66% and correlation coefficient of 0.913. Under current Brazilian conditions, this method generates approximately 60% savings compared to qPCR costs. Conventional RT-PCR, validated herein, showed sufficient results for the detection of SARS-CoV-2 and can be used as an alternative for epidemiological studies and interspecies correlations. Public Library of Science 2021-09-23 /pmc/articles/PMC8459981/ /pubmed/34555073 http://dx.doi.org/10.1371/journal.pone.0257350 Text en © 2021 Carvalho et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Carvalho, Rogério Fernandes
Oliveira, Monike da Silva
Ribeiro, Juliane
dos Santos, Isac Gabriel Cunha
Almeida, Katyane de Sousa
Conti, Ana Carolina Muller
Alexandrino, Bruna
Campos, Fabrício Souza
Soares, Célia Maria de Almeida
Ribeiro Júnior, José Carlos
Validation of conventional PCR-like alternative to SARS-CoV-2 detection with target nucleocapsid protein gene in naso-oropharyngeal samples
title Validation of conventional PCR-like alternative to SARS-CoV-2 detection with target nucleocapsid protein gene in naso-oropharyngeal samples
title_full Validation of conventional PCR-like alternative to SARS-CoV-2 detection with target nucleocapsid protein gene in naso-oropharyngeal samples
title_fullStr Validation of conventional PCR-like alternative to SARS-CoV-2 detection with target nucleocapsid protein gene in naso-oropharyngeal samples
title_full_unstemmed Validation of conventional PCR-like alternative to SARS-CoV-2 detection with target nucleocapsid protein gene in naso-oropharyngeal samples
title_short Validation of conventional PCR-like alternative to SARS-CoV-2 detection with target nucleocapsid protein gene in naso-oropharyngeal samples
title_sort validation of conventional pcr-like alternative to sars-cov-2 detection with target nucleocapsid protein gene in naso-oropharyngeal samples
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8459981/
https://www.ncbi.nlm.nih.gov/pubmed/34555073
http://dx.doi.org/10.1371/journal.pone.0257350
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