Author | Ferreira, Beatriz Iandra da Silva | |
Author | Gomes, Natália Lins da Silva | |
Author | Coelho, Wagner Luis da Costa Nunes Pimentel | |
Author | Costa, Vanessa Duarte da | |
Author | Carneiro, Vanessa Cristine de Souza | |
Author | Kader, Rafael Lopes | |
Author | Amaro, Marisa Pimentel | |
Author | Villar, Lívia Melo | |
Author | Miyajima, Fábio | |
Author | Alves-Leon, Soniza Vieira | |
Author | Paula, Vanessa Salete de | |
Author | Leon, Luciane Almeida Amado | |
Author | Moreira, Otacilio Cruz | |
Access date | 2022-01-11T18:21:22Z | |
Available date | 2022-01-11T18:21:22Z | |
Document date | 2021 | |
Citation | FERREIRA, Beatriz Iandra da Silva et al. Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting. Plos One, v. 16, n. 11, p. 1-17, Nov. 2021. | pt_BR |
ISSN | 1932-6203 | pt_BR |
URI | https://www.arca.fiocruz.br/handle/icict/50731 | |
Language | eng | pt_BR |
Publisher | Public Library of Science | pt_BR |
Rights | open access | pt_BR |
Subject in Portuguese | Validação | pt_BR |
Subject in Portuguese | Ensaio molecular | pt_BR |
Subject in Portuguese | Diagnóstico de COVID-19 | pt_BR |
Subject in Portuguese | Tempor real | pt_BR |
Subject in Portuguese | PCR | pt_BR |
Subject in Portuguese | Fusão de alta resolução | pt_BR |
Title | Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting | pt_BR |
Type | Article | pt_BR |
DOI | 10.1371/journal.pone.0260087 | |
Abstract | The emergence of the COVID-19 pandemic resulted in an unprecedented need for RTqPCR- based molecular diagnostic testing, placing a strain on the supply chain and the availability of commercially available PCR testing kits and reagents. The effect of limited molecular diagnostics-related supplies has been felt across the globe, disproportionally impacting molecular diagnostic testing in developing countries where acquisition of supplies is limited due to availability. The increasing global demand for commercial molecular diagnostic testing kits and reagents has made standard PCR assays cost prohibitive, resulting in the development of alternative approaches to detect SARS-CoV-2 in clinical specimens, circumventing the need for commercial diagnostic testing kits while mitigating the highdemand for molecular diagnostics testing. The timely availability of the complete SARSCoV- 2 genome in the beginning of the COVID-19 pandemic facilitated the rapid development and deployment of specific primers and standardized laboratory protocols for the molecular diagnosis of COVID-19. An alternative method offering a highly specific manner of detecting and genotyping pathogens within clinical specimens is based on the melting temperature differences of PCR products. This method is based on the melting temperature differences between purine and pyrimidine bases. Here, RT-qPCR assays coupled with a High Resolution Melting analysis (HRM-RTqPCR) were developed to target different regions of the SARS-CoV-2 genome (RdRp, E and N) and an internal control (human RNAse P gene). The assays were validated using synthetic sequences from the viral genome and clinical specimens (nasopharyngeal swabs, serum and saliva) of sixty-five patients with severe or moderate COVID-19 from different states within Brazil; a larger validation group than that used in the development to the commercially available TaqMan RTqPCR assay which is considered the gold standard for COVID-19 testing. The sensitivity of the HRM-RTqPCR assays targeting the viral N, RdRp and E genes were 94.12, 98.04 and 92.16%, with 100% specificity to the 3 SARS-CoV-2 genome targets, and a diagnostic accuracy of 95.38, 98.46 and 93.85%, respectively. Thus, HRM-RTqPCR emerges as an attractive alternative and low-cost methodology for the molecular diagnosis of COVID-19 in restricted-budget laboratories. | pt_BR |
Affilliation | Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Real Time PCR Platform RPT09A. Rio de Janeiro, RJ, Brasil. | pt_BR |
Affilliation | Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Real Time PCR Platform RPT09A. Rio de Janeiro, RJ, Brasil. | pt_BR |
Affilliation | Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Desenvolvimento Tecnológico em Virologia. Rio de Janeiro, RJ, Brasil. | pt_BR |
Affilliation | Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hepatites Virais. Rio de Janeiro, RJ, Brasil. | pt_BR |
Affilliation | Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Virologia Molecular. Rio de Janeiro, RJ, Brasil. | pt_BR |
Affilliation | Universidade Federal do Rio de Janeiro. Hospital Universitário Clementino Fraga Filho. Rio de Janeiro, RJ, Brasil. | pt_BR |
Affilliation | Universidade Federal do Rio de Janeiro. Hospital Universitário Clementino Fraga Filho. Rio de Janeiro, RJ, Brasil. | pt_BR |
Affilliation | Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hepatites Virais. Rio de Janeiro, RJ, Brasil. | pt_BR |
Affilliation | Fundação Oswaldo Cruz. Fiocruz Ceará. Eusébio, CE, Brasil. | pt_BR |
Affilliation | Universidade Federal do Rio de Janeiro. Hospital Universitário Clementino Fraga Filho. Rio de Janeiro, RJ, Brasil. | pt_BR |
Affilliation | Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Virologia Molecular. Rio de Janeiro, RJ, Brasil. | pt_BR |
Affilliation | Fundação Oswaldo Cruz. Laboratório de Desenvolvimento Tecnológico em Virologia. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. | pt_BR |
Affilliation | Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Real Time PCR Platform RPT09A. Rio de Janeiro, RJ, Brasil. | pt_BR |
Subject | Validation | pt_BR |
Subject | Molecular assay | pt_BR |
Subject | Diagnostic of COVID-19 | pt_BR |
Subject | Real time PCR | pt_BR |
Subject | High resolution melting | pt_BR |