Development of a Melting-Curve-Based Multiplex Real-Time PCR Assay for the Simultaneous Detection of Viruses Causing Respiratory Infection.

Autor: Tavares ER; Laboratory of Molecular Biology of Microorganisms, Department of Microbiology, State University of Londrina, Londrina 86057-970, Brazil., de Lima TF; Graduate Program in Microbiology, Department of Microbiology, State University of Londrina, Londrina 86057-970, Brazil., Bartolomeu-Gonçalves G; Graduate Program in Clinical and Laboratory Pathophysiology, Department of Pathology, Clinical and Toxicological Analysis, State University of Londrina, Londrina 86038-350, Brazil., de Castro IM; Graduate Program in Microbiology, Department of Microbiology, State University of Londrina, Londrina 86057-970, Brazil., de Lima DG; Laboratory of Molecular Biology of Microorganisms, Department of Microbiology, State University of Londrina, Londrina 86057-970, Brazil., Borges PHG; Graduate Program in Microbiology, Department of Microbiology, State University of Londrina, Londrina 86057-970, Brazil., Nakazato G; Graduate Program in Microbiology, Department of Microbiology, State University of Londrina, Londrina 86057-970, Brazil., Kobayashi RKT; Graduate Program in Microbiology, Department of Microbiology, State University of Londrina, Londrina 86057-970, Brazil., Venancio EJ; Graduate Program in Clinical and Laboratory Pathophysiology, Department of Pathology, Clinical and Toxicological Analysis, State University of Londrina, Londrina 86038-350, Brazil., Tarley CRT; Graduate Program in Chemistry, Department of Chemistry, State University of Londrina, Londrina 86057-970, Brazil., de Almeida ERD; Department of Pathology, Clinical and Toxicological Analysis, State University of Londrina, Londrina 86038-350, Brazil., Pelisson M; Graduate Program in Clinical and Laboratory Pathophysiology, Department of Pathology, Clinical and Toxicological Analysis, State University of Londrina, Londrina 86038-350, Brazil., Vespero EC; Graduate Program in Clinical and Laboratory Pathophysiology, Department of Pathology, Clinical and Toxicological Analysis, State University of Londrina, Londrina 86038-350, Brazil., Simão ANC; Graduate Program in Clinical and Laboratory Pathophysiology, Department of Pathology, Clinical and Toxicological Analysis, State University of Londrina, Londrina 86038-350, Brazil., Perugini MRE; Graduate Program in Clinical and Laboratory Pathophysiology, Department of Pathology, Clinical and Toxicological Analysis, State University of Londrina, Londrina 86038-350, Brazil., Kerbauy G; Graduate Program in Nursing, Department of Nursing, State University of Londrina, Londrina 86038-350, Brazil., Fornazieri MA; Graduate Program in Health Sciences, Department of Clinical Surgery, State University of Londrina, Londrina 86038-350, Brazil., Tognim MCB; Department of Basic Health Sciences, State University of Maringá, Maringá 87020-900, Brazil., Góes VM; Institute of Molecular Biology of Paraná, Curitiba 81350-010, Brazil., Souza TACB; Carlos Chagas Institute, Oswaldo Cruz Foundation (FIOCRUZ-Pr), Curitiba 81350-010, Brazil., Oliveira DBL; Albert Einstein Hospital, São Paulo 05652-900, Brazil.; Laboratory of Clinical and Molecular Virology, University of São Paulo, São Paulo 05508-000, Brazil., Durigon EL; Laboratory of Clinical and Molecular Virology, University of São Paulo, São Paulo 05508-000, Brazil., Faccin-Galhardi LC; Graduate Program in Microbiology, Department of Microbiology, State University of Londrina, Londrina 86057-970, Brazil., Yamauchi LM; Laboratory of Molecular Biology of Microorganisms, Department of Microbiology, State University of Londrina, Londrina 86057-970, Brazil.; Graduate Program in Microbiology, Department of Microbiology, State University of Londrina, Londrina 86057-970, Brazil., Yamada-Ogatta SF; Laboratory of Molecular Biology of Microorganisms, Department of Microbiology, State University of Londrina, Londrina 86057-970, Brazil.; Graduate Program in Microbiology, Department of Microbiology, State University of Londrina, Londrina 86057-970, Brazil.
Jazyk: angličtina
Zdroj: Microorganisms [Microorganisms] 2023 Nov 02; Vol. 11 (11). Date of Electronic Publication: 2023 Nov 02.
DOI: 10.3390/microorganisms11112692
Abstrakt: 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.
Databáze: MEDLINE