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

被引：4

作者：

Tavares, Eliandro Reis ^{[1
]}

de Lima, Thiago Ferreira ^{[2
]}

Bartolomeu-Goncalves, Guilherme ^{[3
]}

de Castro, Isabela Madeira ^{[2
]}

de Lima, Daniel Gaiotto ^{[1
]}

Borges, Paulo Henrique Guilherme ^{[2
]}

Nakazato, Gerson ^{[2
]}

Kobayashi, Renata Katsuko Takayama ^{[2
]}

Venancio, Emerson Jose ^{[3
]}

Tarley, Cesar Ricardo Teixeira ^{[4
]}

de Almeida, Elaine Regina Delicato ^{[5
]}

Pelisson, Marsileni ^{[3
]}

Vespero, Eliana Carolina ^{[3
]}

Simao, Andrea Name Colado ^{[3
]}

Perugini, Marcia Regina Eches ^{[3
]}

Kerbauy, Gilselena ^{[6
]}

Fornazieri, Marco Aurelio ^{[7
]}

Tognim, Maria Cristina Bronharo ^{[8
]}

Goes, Viviane Monteiro ^{[9
]}

Souza, Tatiana de Arruda Campos Brasil de ^{[10
]}

Oliveira, Danielle Bruna Leal ^{[11
,12
]}

Durigon, Edison Luiz ^{[12
]}

Faccin-Galhardi, Ligia Carla ^{[2
]}

Yamauchi, Lucy Megumi ^{[1
,2
]}

Yamada-Ogatta, Sueli Fumie ^{[1
,2
]}

机构：

[1] Univ Estadual Londrina, Dept Microbiol, Lab Mol Biol Microorganisms, BR-86057970 Londrina, Brazil

[2] Univ Estadual Londrina, Dept Microbiol, Grad Program Microbiol, BR-86057970 Londrina, Brazil

[3] Univ Estadual Londrina, Dept Pathol, Grad Program, Clin & Lab Pathophysiol, BR-86038350 Londrina, Brazil

[4] Univ Estadual Londrina, Dept Chem, Grad Program Chem, BR-86057970 Londrina, Brazil

[5] Univ Estadual Londrina, Dept Pathol Clin & Toxicol Anal, BR-86038350 Londrina, Brazil

[6] Univ Estadual Londrina, Dept Nursing, Grad Program Nursing, BR-86038350 Londrina, Brazil

[7] Univ Estadual Londrina, Dept Clin Surg, Grad Program Hlth Sci, BR-86038350 Londrina, Brazil

[8] Univ Estadual Maringa, Dept Basic Hlth Sci, BR-87020900 Maringa, Brazil

[9] Inst Mol Biol Parana, BR-81350010 Curitiba, Brazil

[10] Oswaldo Cruz Fdn FIOCRUZ Pr, Carlos Chagas Inst, BR-81350010 Curitiba, Brazil

[11] Albert Einstein Hosp, BR-05652900 Sao Paulo, Brazil

[12] Univ Sao Paulo, Lab Clin & Mol Virol, BR-05508000 Sao Paulo, Brazil

来源：

MICROORGANISMS | 2023年 / 11卷 / 11期

关键词：

COVID-19; SARS-CoV-2; Influenza A virus; Human Respiratory Syncytial Virus; Human Rhinovirus B; nucleic acid amplification test; SARS-COV-2; INFLUENZA;

D O I：

10.3390/microorganisms11112692

中图分类号：

Q93 [微生物学];

学科分类号：

071005 ; 100705 ;

摘要：

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.

引用

页数：16

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