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Duplex On-Site Detection of Vibrio cholerae and Vibrio vulnificus by Recombinase Polymerase Amplification and Three-Segment Lateral Flow Strips
Vibrio cholerae and Vibrio vulnificus are two most reported foodborne Vibrio pathogens related to seafood. Due to global ocean warming and an increase in seafood consumption worldwide, foodborne illnesses related to infection of these two bacteria are growing, leading to food safety issues and econo...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8151630/ https://www.ncbi.nlm.nih.gov/pubmed/34066017 http://dx.doi.org/10.3390/bios11050151 |
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author | Wang, Pei Liao, Lei Ma, Chao Zhang, Xue Yu, Junwei Yi, Longyu Liu, Xin Shen, Hui Gao, Song Lu, Qunwei |
author_facet | Wang, Pei Liao, Lei Ma, Chao Zhang, Xue Yu, Junwei Yi, Longyu Liu, Xin Shen, Hui Gao, Song Lu, Qunwei |
author_sort | Wang, Pei |
collection | PubMed |
description | Vibrio cholerae and Vibrio vulnificus are two most reported foodborne Vibrio pathogens related to seafood. Due to global ocean warming and an increase in seafood consumption worldwide, foodborne illnesses related to infection of these two bacteria are growing, leading to food safety issues and economic consequences. Molecular detection methods targeting species-specific genes are effective tools in the fight against bacterial infections for food safety. In this study, a duplex detection biosensor based on isothermal recombinase polymerase amplification (RPA) and a three-segment lateral flow strip (LFS) has been established. The biosensor used lolB gene of Vibrio cholerae and empV gene of Vibrio vulnificus as the detection markers based on previous reports. A duplex RPA reaction for both targets were constructed, and two chemical labels, FITC and DIG, of the amplification products were carefully tested for effective and accurate visualization on the strip. The biosensor demonstrated good specificity and achieved a sensitivity of 10(1) copies per reaction or one colony forming unit (CFU)/10 g of spiked food for both bacteria. Validation with clinical samples showed results consistent with that of real-time polymerase chain reaction. The detection process was simple and fast with a 30-min reaction at 37 °C and visualization on the strip within 5 min. With little dependence on laboratory settings, this biosensor was suitable for on-site detection, and the duplex system enabled simultaneous detection of the two important foodborne bacteria. Moreover, the principle can be extended to healthcare and food safety applications for other pathogens. |
format | Online Article Text |
id | pubmed-8151630 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-81516302021-05-27 Duplex On-Site Detection of Vibrio cholerae and Vibrio vulnificus by Recombinase Polymerase Amplification and Three-Segment Lateral Flow Strips Wang, Pei Liao, Lei Ma, Chao Zhang, Xue Yu, Junwei Yi, Longyu Liu, Xin Shen, Hui Gao, Song Lu, Qunwei Biosensors (Basel) Article Vibrio cholerae and Vibrio vulnificus are two most reported foodborne Vibrio pathogens related to seafood. Due to global ocean warming and an increase in seafood consumption worldwide, foodborne illnesses related to infection of these two bacteria are growing, leading to food safety issues and economic consequences. Molecular detection methods targeting species-specific genes are effective tools in the fight against bacterial infections for food safety. In this study, a duplex detection biosensor based on isothermal recombinase polymerase amplification (RPA) and a three-segment lateral flow strip (LFS) has been established. The biosensor used lolB gene of Vibrio cholerae and empV gene of Vibrio vulnificus as the detection markers based on previous reports. A duplex RPA reaction for both targets were constructed, and two chemical labels, FITC and DIG, of the amplification products were carefully tested for effective and accurate visualization on the strip. The biosensor demonstrated good specificity and achieved a sensitivity of 10(1) copies per reaction or one colony forming unit (CFU)/10 g of spiked food for both bacteria. Validation with clinical samples showed results consistent with that of real-time polymerase chain reaction. The detection process was simple and fast with a 30-min reaction at 37 °C and visualization on the strip within 5 min. With little dependence on laboratory settings, this biosensor was suitable for on-site detection, and the duplex system enabled simultaneous detection of the two important foodborne bacteria. Moreover, the principle can be extended to healthcare and food safety applications for other pathogens. MDPI 2021-05-12 /pmc/articles/PMC8151630/ /pubmed/34066017 http://dx.doi.org/10.3390/bios11050151 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Wang, Pei Liao, Lei Ma, Chao Zhang, Xue Yu, Junwei Yi, Longyu Liu, Xin Shen, Hui Gao, Song Lu, Qunwei Duplex On-Site Detection of Vibrio cholerae and Vibrio vulnificus by Recombinase Polymerase Amplification and Three-Segment Lateral Flow Strips |
title | Duplex On-Site Detection of Vibrio cholerae and Vibrio vulnificus by Recombinase Polymerase Amplification and Three-Segment Lateral Flow Strips |
title_full | Duplex On-Site Detection of Vibrio cholerae and Vibrio vulnificus by Recombinase Polymerase Amplification and Three-Segment Lateral Flow Strips |
title_fullStr | Duplex On-Site Detection of Vibrio cholerae and Vibrio vulnificus by Recombinase Polymerase Amplification and Three-Segment Lateral Flow Strips |
title_full_unstemmed | Duplex On-Site Detection of Vibrio cholerae and Vibrio vulnificus by Recombinase Polymerase Amplification and Three-Segment Lateral Flow Strips |
title_short | Duplex On-Site Detection of Vibrio cholerae and Vibrio vulnificus by Recombinase Polymerase Amplification and Three-Segment Lateral Flow Strips |
title_sort | duplex on-site detection of vibrio cholerae and vibrio vulnificus by recombinase polymerase amplification and three-segment lateral flow strips |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8151630/ https://www.ncbi.nlm.nih.gov/pubmed/34066017 http://dx.doi.org/10.3390/bios11050151 |
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