Molecular Beacon Assay Development for Severe Acute Respiratory Syndrome Coronavirus 2 Detection
The fast spread of SARS-CoV-2 has led to a global pandemic, calling for fast and accurate assays to allow infection diagnosis and prevention of transmission. We aimed to develop a molecular beacon (MB)-based detection assay for SARS-CoV-2, designed to detect the ORF1ab and S genes, proposing a two-s...
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Published in | Sensors (Basel, Switzerland) Vol. 21; no. 21; p. 7015 |
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Main Authors | , , , , , , , , , , , , , |
Format | Journal Article |
Language | English |
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22.10.2021
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Abstract | The fast spread of SARS-CoV-2 has led to a global pandemic, calling for fast and accurate assays to allow infection diagnosis and prevention of transmission. We aimed to develop a molecular beacon (MB)-based detection assay for SARS-CoV-2, designed to detect the ORF1ab and S genes, proposing a two-stage COVID-19 testing strategy. The novelty of this work lies in the design and optimization of two MBs for detection of SARS-CoV-2, namely, concentration, fluorescence plateaus of hybridization, reaction temperature and real-time results. We also identify putative G-quadruplex (G4) regions in the genome of SARS-CoV-2. A total of 458 nasopharyngeal and throat swab samples (426 positive and 32 negative) were tested with the MB assay and the fluorescence levels compared with the cycle threshold (Ct) values obtained from a commercial RT-PCR test in terms of test duration, sensitivity, and specificity. Our results show that the samples with higher fluorescence levels correspond to those with low Ct values, suggesting a correlation between viral load and increased MB fluorescence. The proposed assay represents a fast (total duration of 2 h 20 min including amplification and fluorescence reading stages) and simple way of detecting SARS-CoV-2 in clinical samples from the upper respiratory tract. |
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AbstractList | The fast spread of SARS-CoV-2 has led to a global pandemic, calling for fast and accurate assays to allow infection diagnosis and prevention of transmission. We aimed to develop a molecular beacon (MB)-based detection assay for SARS-CoV-2, designed to detect the ORF1ab and S genes, proposing a two-stage COVID-19 testing strategy. The novelty of this work lies in the design and optimization of two MBs for detection of SARS-CoV-2, namely, concentration, fluorescence plateaus of hybridization, reaction temperature and real-time results. We also identify putative G-quadruplex (G4) regions in the genome of SARS-CoV-2. A total of 458 nasopharyngeal and throat swab samples (426 positive and 32 negative) were tested with the MB assay and the fluorescence levels compared with the cycle threshold (Ct) values obtained from a commercial RT-PCR test in terms of test duration, sensitivity, and specificity. Our results show that the samples with higher fluorescence levels correspond to those with low Ct values, suggesting a correlation between viral load and increased MB fluorescence. The proposed assay represents a fast (total duration of 2 h 20 min including amplification and fluorescence reading stages) and simple way of detecting SARS-CoV-2 in clinical samples from the upper respiratory tract. |
Author | Santos, Tiago Carvalho, Josué Socorro, Sílvia Lopes-Nunes, Jéssica Teixeira, Rui Sousa, Fani Cruz, Carla Faria, Conceição Figueiredo, Joana Duarte, Ana Paula Tomaz, Cândida Teixeira Miranda, André Felgueiras, Mafalda Riscado, Micaela |
AuthorAffiliation | 3 Serviço de Patologia Clínica do Centro Hospitalar Universitário Cova da Beira (CHUCB), 6200-251 Covilhã, Portugal; mafalda.felgueiras@ulsm.min-saude.pt (M.F.); rteixeira@chcbeira.min-saude.pt (R.T.); cfaria@chcbeira.min-saude.pt (C.F.) 1 CICS-UBI—Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal; josue.carvalho@fcsaude.ubi.pt (J.C.); jessica.nunes@ubi.pt (J.L.-N.); joana.figueiredo@ubi.pt (J.F.); tiago.santos@fcsaude.ubi.pt (T.S.); andre.miranda@ubi.pt (A.M.); micaela.riscado@ubi.pt (M.R.); fani.sousa@fcsaude.ubi.pt (F.S.); apduarte@fcsaude.ubi.pt (A.P.D.); ssocorro@fcsaude.ubi.pt (S.S.); ctomaz@ubi.pt (C.T.T.) 2 C4—Cloud Computing Competence Centre, UBIMedical, Universidade da Beira Interior, Estrada Municipal 506, 6200-284 Covilhã, Portugal |
AuthorAffiliation_xml | – name: 2 C4—Cloud Computing Competence Centre, UBIMedical, Universidade da Beira Interior, Estrada Municipal 506, 6200-284 Covilhã, Portugal – name: 3 Serviço de Patologia Clínica do Centro Hospitalar Universitário Cova da Beira (CHUCB), 6200-251 Covilhã, Portugal; mafalda.felgueiras@ulsm.min-saude.pt (M.F.); rteixeira@chcbeira.min-saude.pt (R.T.); cfaria@chcbeira.min-saude.pt (C.F.) – name: 1 CICS-UBI—Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal; josue.carvalho@fcsaude.ubi.pt (J.C.); jessica.nunes@ubi.pt (J.L.-N.); joana.figueiredo@ubi.pt (J.F.); tiago.santos@fcsaude.ubi.pt (T.S.); andre.miranda@ubi.pt (A.M.); micaela.riscado@ubi.pt (M.R.); fani.sousa@fcsaude.ubi.pt (F.S.); apduarte@fcsaude.ubi.pt (A.P.D.); ssocorro@fcsaude.ubi.pt (S.S.); ctomaz@ubi.pt (C.T.T.) |
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SubjectTerms | Assaying Bioinformatics Coronaviruses COVID-19 COVID-19 Testing CRISPR Design optimization diagnosis Disease transmission Fluorescence fluorescence detection Genomes Humans Medical diagnosis NMR Nuclear magnetic resonance Pandemics Proteins Respiratory diseases RNA viruses RNA, Viral SARS-CoV-2 Sensitivity and Specificity Severe acute respiratory syndrome coronavirus 2 Spectrum analysis Thermal cycling Viral diseases Viruses |
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Title | Molecular Beacon Assay Development for Severe Acute Respiratory Syndrome Coronavirus 2 Detection |
URI | https://www.ncbi.nlm.nih.gov/pubmed/34770321 https://www.proquest.com/docview/2596069720/abstract/ https://search.proquest.com/docview/2597484749 https://pubmed.ncbi.nlm.nih.gov/PMC8587319 https://doaj.org/article/46073fa94116449d9743c9201aa73dd0 |
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