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

• Published in: PloS one Vol. 16; no. 11; p. e0260087
• Main Authors: Ferreira, Beatriz Iandra da Silva, da Silva-Gomes, Natália Lins, Coelho, Wagner Luis da Costa Nunes Pimentel, da Costa, Vanessa Duarte, Carneiro, Vanessa Cristine de Souza, Kader, Rafael Lopes, Amaro, Marisa Pimentel, Villar, Lívia Melo, Miyajima, Fábio, Alves-Leon, Soniza Vieira, de Paula, Vanessa Salete, Leon, Luciane Almeida Amado, Moreira, Otacilio Cruz
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 22.11.2021; Public Library of Science (PLoS)
• Subjects: Adult; Assaying; Availability; Biochemical assays; Biology and life sciences; Coronaviruses; COVID-19; COVID-19 Nucleic Acid Testing - methods; COVID-19 Nucleic Acid Testing - standards; Developing countries; Diagnosis; Diagnostic systems; Disease transmission; Female; Genomes; Genotyping; Hepatitis; High resolution; Hospitals; Humans; LDCs; Male; Medical diagnosis; Medical laboratories; Medicine and health sciences; Melt temperature; Melting; Methods; Molecular biology; Molecular diagnostic techniques; Nucleic Acid Denaturation; Oligonucleotides - chemistry; Pandemics; Pathogens; Patients; Physical Sciences; Polymerase chain reaction; Reagents; Real time; Real-Time Polymerase Chain Reaction - methods; Real-Time Polymerase Chain Reaction - standards; Research and Analysis Methods; Respiratory Mucosa - virology; Ribonuclease P; RNA polymerase; Saliva; Saliva - virology; SARS-CoV-2 - genetics; SARS-CoV-2 - pathogenicity; Sensitivity analysis; Sensitivity and Specificity; Severe acute respiratory syndrome; Severe acute respiratory syndrome coronavirus 2; Supply chains; Temperature gradients; Viral diseases; Virology; Brazil; Rio de Janeiro Brazil; United States > US; Germany
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0260087

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.