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Development and validation of a PCR-free nucleic acid testing method for RNA viruses based on linear molecular beacon probes

BACKGROUND: RNA viruses periodically trigger pandemics of severe human diseases, frequently causing enormous economic losses. Here, a nucleic acid extraction-free and amplification-free RNA virus testing probe was proposed for the sensitive and simple detection of classical swine fever virus (CSFV)...

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Autores principales: Du, Fuyu, Zhang, Weijie, Yao, Huimin, Xia, Yuqiong, Zhang, Xianghan, Yang, Peng, Ning, Pengbo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9187886/
https://www.ncbi.nlm.nih.gov/pubmed/35690818
http://dx.doi.org/10.1186/s12951-022-01470-1
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author Du, Fuyu
Zhang, Weijie
Yao, Huimin
Xia, Yuqiong
Zhang, Xianghan
Yang, Peng
Ning, Pengbo
author_facet Du, Fuyu
Zhang, Weijie
Yao, Huimin
Xia, Yuqiong
Zhang, Xianghan
Yang, Peng
Ning, Pengbo
author_sort Du, Fuyu
collection PubMed
description BACKGROUND: RNA viruses periodically trigger pandemics of severe human diseases, frequently causing enormous economic losses. Here, a nucleic acid extraction-free and amplification-free RNA virus testing probe was proposed for the sensitive and simple detection of classical swine fever virus (CSFV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), based on a double-stranded molecular beacon method. This RNA virus probe contains two base sequences—a recognition strand that binds to the specific domain of CSFV N2 or SARS-CoV-2 N, with a fluorophore (FAM) labeled at the 5′ end, and a complementary strand (CSFV-Probe B or SARS-CoV-2-Probe B), combined with a quencher (BHQ2) labeled at the 3′ end. RESULTS: Using linear molecular beacon probe technology, the detection limit of the RNA virus probe corresponding to CSFV and SARS-CoV-2 were as low as 0.28 nM and 0.24 nM, respectively. After CSFV E2 and SARS-CoV-2 N genes were transfected into corresponding host cells, the monitoring of RNA virus probes showed that fluorescence signals were dramatically enhanced in a concentration- and time-dependent manner. These results were supported by those of quantitative (qRT-PCR) and visualization (confocal microscopy) analyses. Furthermore, CSF-positive swine samples and simulated SARS-CoV-2 infected mouse samples were used to demonstrate their applicability for different distributions of viral nucleic acids in series tissues. CONCLUSIONS: The proposed RNA virus probe could be used as a PCR-free, cost-effective, and rapid point-of-care (POC) diagnostic platform for target RNA virus detection, holding great potential for the convenient monitoring of different RNA viruses for early mass virus screening. GRAPHICAL ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12951-022-01470-1.
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spelling pubmed-91878862022-06-12 Development and validation of a PCR-free nucleic acid testing method for RNA viruses based on linear molecular beacon probes Du, Fuyu Zhang, Weijie Yao, Huimin Xia, Yuqiong Zhang, Xianghan Yang, Peng Ning, Pengbo J Nanobiotechnology Research BACKGROUND: RNA viruses periodically trigger pandemics of severe human diseases, frequently causing enormous economic losses. Here, a nucleic acid extraction-free and amplification-free RNA virus testing probe was proposed for the sensitive and simple detection of classical swine fever virus (CSFV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), based on a double-stranded molecular beacon method. This RNA virus probe contains two base sequences—a recognition strand that binds to the specific domain of CSFV N2 or SARS-CoV-2 N, with a fluorophore (FAM) labeled at the 5′ end, and a complementary strand (CSFV-Probe B or SARS-CoV-2-Probe B), combined with a quencher (BHQ2) labeled at the 3′ end. RESULTS: Using linear molecular beacon probe technology, the detection limit of the RNA virus probe corresponding to CSFV and SARS-CoV-2 were as low as 0.28 nM and 0.24 nM, respectively. After CSFV E2 and SARS-CoV-2 N genes were transfected into corresponding host cells, the monitoring of RNA virus probes showed that fluorescence signals were dramatically enhanced in a concentration- and time-dependent manner. These results were supported by those of quantitative (qRT-PCR) and visualization (confocal microscopy) analyses. Furthermore, CSF-positive swine samples and simulated SARS-CoV-2 infected mouse samples were used to demonstrate their applicability for different distributions of viral nucleic acids in series tissues. CONCLUSIONS: The proposed RNA virus probe could be used as a PCR-free, cost-effective, and rapid point-of-care (POC) diagnostic platform for target RNA virus detection, holding great potential for the convenient monitoring of different RNA viruses for early mass virus screening. GRAPHICAL ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12951-022-01470-1. BioMed Central 2022-06-11 /pmc/articles/PMC9187886/ /pubmed/35690818 http://dx.doi.org/10.1186/s12951-022-01470-1 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Research
Du, Fuyu
Zhang, Weijie
Yao, Huimin
Xia, Yuqiong
Zhang, Xianghan
Yang, Peng
Ning, Pengbo
Development and validation of a PCR-free nucleic acid testing method for RNA viruses based on linear molecular beacon probes
title Development and validation of a PCR-free nucleic acid testing method for RNA viruses based on linear molecular beacon probes
title_full Development and validation of a PCR-free nucleic acid testing method for RNA viruses based on linear molecular beacon probes
title_fullStr Development and validation of a PCR-free nucleic acid testing method for RNA viruses based on linear molecular beacon probes
title_full_unstemmed Development and validation of a PCR-free nucleic acid testing method for RNA viruses based on linear molecular beacon probes
title_short Development and validation of a PCR-free nucleic acid testing method for RNA viruses based on linear molecular beacon probes
title_sort development and validation of a pcr-free nucleic acid testing method for rna viruses based on linear molecular beacon probes
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9187886/
https://www.ncbi.nlm.nih.gov/pubmed/35690818
http://dx.doi.org/10.1186/s12951-022-01470-1
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