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- Title
Development of a Melting-Curve-Based Multiplex Real-Time PCR Assay for the Simultaneous Detection of Viruses Causing Respiratory Infection.
- Authors
Tavares, Eliandro Reis; de Lima, Thiago Ferreira; Bartolomeu-Gonçalves, Guilherme; de Castro, Isabela Madeira; de Lima, Daniel Gaiotto; Borges, Paulo Henrique Guilherme; Nakazato, Gerson; Kobayashi, Renata Katsuko Takayama; Venancio, Emerson José; Tarley, César Ricardo Teixeira; de Almeida, Elaine Regina Delicato; Pelisson, Marsileni; Vespero, Eliana Carolina; Simão, Andrea Name Colado; Perugini, Márcia Regina Eches; Kerbauy, Gilselena; Fornazieri, Marco Aurélio; Tognim, Maria Cristina Bronharo; Góes, Viviane Monteiro; Souza, Tatiana de Arruda Campos Brasil de
- Abstract
The prompt and accurate identification of the etiological agents of viral respiratory infections is a critical measure in mitigating outbreaks. In this study, we developed and clinically evaluated a novel melting-curve-based multiplex real-time PCR (M-m-qPCR) assay targeting the RNA-dependent RNA polymerase (RdRp) and nucleocapsid phosphoprotein N of SARS-CoV-2, the Matrix protein 2 of the Influenza A virus, the RdRp domain of the L protein from the Human Respiratory Syncytial Virus, and the polyprotein from Rhinovirus B genes. The analytical performance of the M-m-qPCR underwent assessment using in silico analysis and a panel of reference and clinical strains, encompassing viral, bacterial, and fungal pathogens, exhibiting 100% specificity. Moreover, the assay showed a detection limit of 10 copies per reaction for all targeted pathogens using the positive controls. To validate its applicability, the assay was further tested in simulated nasal fluid spiked with the viruses mentioned above, followed by validation on nasopharyngeal swabs collected from 811 individuals. Among them, 13.4% (109/811) tested positive for SARS-CoV-2, and 1.1% (9/811) tested positive for Influenza A. Notably, these results showed 100% concordance with those obtained using a commercial kit. Therefore, the M-m-qPCR exhibits great potential for the routine screening of these respiratory viral pathogens.
- Subjects
RESPIRATORY infections; RESPIRATORY syncytial virus; RNA replicase; VIRUS diseases; INFLUENZA viruses
- Publication
Microorganisms, 2023, Vol 11, Issue 11, p2692
- ISSN
2076-2607
- Publication type
Article
- DOI
10.3390/microorganisms11112692