Development of a Melting-Curve-Based Multiplex Real-Time PCR Assay for the Simultaneous Detection of Viruses Causing Respiratory Infection
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 po...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-11-01
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| Series: | Microorganisms |
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| Online Access: | https://www.mdpi.com/2076-2607/11/11/2692 |
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| author | 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 |
| author_facet | 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 |
| author_sort | Eliandro Reis Tavares |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-09T16:35:45Z |
| format | Article |
| id | doaj.art-f12151929c37496c98c26c6b8b645d85 |
| institution | Directory Open Access Journal |
| issn | 2076-2607 |
| language | English |
| last_indexed | 2024-03-09T16:35:45Z |
| publishDate | 2023-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Microorganisms |
| spelling | doaj.art-f12151929c37496c98c26c6b8b645d852023-11-24T14:56:56ZengMDPI AGMicroorganisms2076-26072023-11-011111269210.3390/microorganisms11112692Development of a Melting-Curve-Based Multiplex Real-Time PCR Assay for the Simultaneous Detection of Viruses Causing Respiratory InfectionEliandro Reis Tavares0Thiago Ferreira de Lima1Guilherme Bartolomeu-Gonçalves2Isabela Madeira de Castro3Daniel Gaiotto de Lima4Paulo Henrique Guilherme Borges5Gerson Nakazato6Renata Katsuko Takayama Kobayashi7Emerson José Venancio8César Ricardo Teixeira Tarley9Elaine Regina Delicato de Almeida10Marsileni Pelisson11Eliana Carolina Vespero12Andrea Name Colado Simão13Márcia Regina Eches Perugini14Gilselena Kerbauy15Marco Aurélio Fornazieri16Maria Cristina Bronharo Tognim17Viviane Monteiro Góes18Tatiana de Arruda Campos Brasil de Souza19Danielle Bruna Leal Oliveira20Edison Luiz Durigon21Lígia Carla Faccin-Galhardi22Lucy Megumi Yamauchi23Sueli Fumie Yamada-Ogatta24Laboratory of Molecular Biology of Microorganisms, Department of Microbiology, State University of Londrina, Londrina 86057-970, BrazilGraduate Program in Microbiology, Department of Microbiology, State University of Londrina, Londrina 86057-970, BrazilGraduate Program in Clinical and Laboratory Pathophysiology, Department of Pathology, Clinical and Toxicological Analysis, State University of Londrina, Londrina 86038-350, BrazilGraduate Program in Microbiology, Department of Microbiology, State University of Londrina, Londrina 86057-970, BrazilLaboratory of Molecular Biology of Microorganisms, Department of Microbiology, State University of Londrina, Londrina 86057-970, BrazilGraduate Program in Microbiology, Department of Microbiology, State University of Londrina, Londrina 86057-970, BrazilGraduate Program in Microbiology, Department of Microbiology, State University of Londrina, Londrina 86057-970, BrazilGraduate Program in Microbiology, Department of Microbiology, State University of Londrina, Londrina 86057-970, BrazilGraduate Program in Clinical and Laboratory Pathophysiology, Department of Pathology, Clinical and Toxicological Analysis, State University of Londrina, Londrina 86038-350, BrazilGraduate Program in Chemistry, Department of Chemistry, State University of Londrina, Londrina 86057-970, BrazilDepartment of Pathology, Clinical and Toxicological Analysis, State University of Londrina, Londrina 86038-350, BrazilGraduate Program in Clinical and Laboratory Pathophysiology, Department of Pathology, Clinical and Toxicological Analysis, State University of Londrina, Londrina 86038-350, BrazilGraduate Program in Clinical and Laboratory Pathophysiology, Department of Pathology, Clinical and Toxicological Analysis, State University of Londrina, Londrina 86038-350, BrazilGraduate Program in Clinical and Laboratory Pathophysiology, Department of Pathology, Clinical and Toxicological Analysis, State University of Londrina, Londrina 86038-350, BrazilGraduate Program in Clinical and Laboratory Pathophysiology, Department of Pathology, Clinical and Toxicological Analysis, State University of Londrina, Londrina 86038-350, BrazilGraduate Program in Nursing, Department of Nursing, State University of Londrina, Londrina 86038-350, BrazilGraduate Program in Health Sciences, Department of Clinical Surgery, State University of Londrina, Londrina 86038-350, BrazilDepartment of Basic Health Sciences, State University of Maringá, Maringá 87020-900, BrazilInstitute of Molecular Biology of Paraná, Curitiba 81350-010, BrazilCarlos Chagas Institute, Oswaldo Cruz Foundation (FIOCRUZ-Pr), Curitiba 81350-010, BrazilAlbert Einstein Hospital, São Paulo 05652-900, BrazilLaboratory of Clinical and Molecular Virology, University of São Paulo, São Paulo 05508-000, BrazilGraduate Program in Microbiology, Department of Microbiology, State University of Londrina, Londrina 86057-970, BrazilLaboratory of Molecular Biology of Microorganisms, Department of Microbiology, State University of Londrina, Londrina 86057-970, BrazilLaboratory of Molecular Biology of Microorganisms, Department of Microbiology, State University of Londrina, Londrina 86057-970, BrazilThe 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.https://www.mdpi.com/2076-2607/11/11/2692COVID-19SARS-CoV-2Influenza A virusHuman Respiratory Syncytial VirusHuman Rhinovirus Bnucleic acid amplification test |
| spellingShingle | 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 Development of a Melting-Curve-Based Multiplex Real-Time PCR Assay for the Simultaneous Detection of Viruses Causing Respiratory Infection Microorganisms COVID-19 SARS-CoV-2 Influenza A virus Human Respiratory Syncytial Virus Human Rhinovirus B nucleic acid amplification test |
| title | Development of a Melting-Curve-Based Multiplex Real-Time PCR Assay for the Simultaneous Detection of Viruses Causing Respiratory Infection |
| title_full | Development of a Melting-Curve-Based Multiplex Real-Time PCR Assay for the Simultaneous Detection of Viruses Causing Respiratory Infection |
| title_fullStr | Development of a Melting-Curve-Based Multiplex Real-Time PCR Assay for the Simultaneous Detection of Viruses Causing Respiratory Infection |
| title_full_unstemmed | Development of a Melting-Curve-Based Multiplex Real-Time PCR Assay for the Simultaneous Detection of Viruses Causing Respiratory Infection |
| title_short | Development of a Melting-Curve-Based Multiplex Real-Time PCR Assay for the Simultaneous Detection of Viruses Causing Respiratory Infection |
| title_sort | development of a melting curve based multiplex real time pcr assay for the simultaneous detection of viruses causing respiratory infection |
| topic | COVID-19 SARS-CoV-2 Influenza A virus Human Respiratory Syncytial Virus Human Rhinovirus B nucleic acid amplification test |
| url | https://www.mdpi.com/2076-2607/11/11/2692 |
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