Microorganisms (Nov 2023)

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

  • Eliandro Reis Tavares,
  • Thiago Ferreira de Lima,
  • Guilherme Bartolomeu-Gonçalves,
  • Isabela Madeira de Castro,
  • Daniel Gaiotto de Lima,
  • Paulo Henrique Guilherme Borges,
  • Gerson Nakazato,
  • Renata Katsuko Takayama Kobayashi,
  • Emerson José Venancio,
  • César Ricardo Teixeira Tarley,
  • Elaine Regina Delicato de Almeida,
  • Marsileni Pelisson,
  • Eliana Carolina Vespero,
  • Andrea Name Colado Simão,
  • Márcia Regina Eches Perugini,
  • Gilselena Kerbauy,
  • Marco Aurélio Fornazieri,
  • Maria Cristina Bronharo Tognim,
  • Viviane Monteiro Góes,
  • Tatiana de Arruda Campos Brasil de Souza,
  • Danielle Bruna Leal Oliveira,
  • Edison Luiz Durigon,
  • Lígia Carla Faccin-Galhardi,
  • Lucy Megumi Yamauchi,
  • Sueli Fumie Yamada-Ogatta

DOI
https://doi.org/10.3390/microorganisms11112692
Journal volume & issue
Vol. 11, no. 11
p. 2692

Abstract

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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.

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