Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting.

The emergence of the COVID-19 pandemic resulted in an unprecedented need for RT-qPCR-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...

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Autores principales:	Beatriz Iandra da Silva Ferreira, Natália Lins da Silva-Gomes, Wagner Luis da Costa Nunes Pimentel Coelho, Vanessa Duarte da Costa, Vanessa Cristine de Souza Carneiro, Rafael Lopes Kader, Marisa Pimentel Amaro, Lívia Melo Villar, Fábio Miyajima, Soniza Vieira Alves-Leon, Vanessa Salete de Paula, Luciane Almeida Amado Leon, Otacilio Cruz Moreira
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/f996708d4ad944bd97eb3dc6d227fea8
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spelling	oai:doaj.org-article:f996708d4ad944bd97eb3dc6d227fea82021-12-02T20:16:19ZValidation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting.1932-620310.1371/journal.pone.0260087https://doaj.org/article/f996708d4ad944bd97eb3dc6d227fea82021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0260087https://doaj.org/toc/1932-6203The emergence of the COVID-19 pandemic resulted in an unprecedented need for RT-qPCR-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 high-demand for molecular diagnostics testing. The timely availability of the complete SARS-CoV-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 RT-qPCR 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.Beatriz Iandra da Silva FerreiraNatália Lins da Silva-GomesWagner Luis da Costa Nunes Pimentel CoelhoVanessa Duarte da CostaVanessa Cristine de Souza CarneiroRafael Lopes KaderMarisa Pimentel AmaroLívia Melo VillarFábio MiyajimaSoniza Vieira Alves-LeonVanessa Salete de PaulaLuciane Almeida Amado LeonOtacilio Cruz MoreiraPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 11, p e0260087 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Beatriz Iandra da Silva Ferreira Natália Lins da Silva-Gomes Wagner Luis da Costa Nunes Pimentel Coelho Vanessa Duarte da Costa Vanessa Cristine de Souza Carneiro Rafael Lopes Kader Marisa Pimentel Amaro Lívia Melo Villar Fábio Miyajima Soniza Vieira Alves-Leon Vanessa Salete de Paula Luciane Almeida Amado Leon Otacilio Cruz Moreira Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting.
description	The emergence of the COVID-19 pandemic resulted in an unprecedented need for RT-qPCR-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 high-demand for molecular diagnostics testing. The timely availability of the complete SARS-CoV-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 RT-qPCR 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.
format	article
author	Beatriz Iandra da Silva Ferreira Natália Lins da Silva-Gomes Wagner Luis da Costa Nunes Pimentel Coelho Vanessa Duarte da Costa Vanessa Cristine de Souza Carneiro Rafael Lopes Kader Marisa Pimentel Amaro Lívia Melo Villar Fábio Miyajima Soniza Vieira Alves-Leon Vanessa Salete de Paula Luciane Almeida Amado Leon Otacilio Cruz Moreira
author_facet	Beatriz Iandra da Silva Ferreira Natália Lins da Silva-Gomes Wagner Luis da Costa Nunes Pimentel Coelho Vanessa Duarte da Costa Vanessa Cristine de Souza Carneiro Rafael Lopes Kader Marisa Pimentel Amaro Lívia Melo Villar Fábio Miyajima Soniza Vieira Alves-Leon Vanessa Salete de Paula Luciane Almeida Amado Leon Otacilio Cruz Moreira
author_sort	Beatriz Iandra da Silva Ferreira
title	Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting.
title_short	Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting.
title_full	Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting.
title_fullStr	Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting.
title_full_unstemmed	Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting.
title_sort	validation of a novel molecular assay to the diagnostic of covid-19 based on real time pcr with high resolution melting.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/f996708d4ad944bd97eb3dc6d227fea8
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Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting.

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