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A Lab-on-a-Tube Biosensor Combining Recombinase-Aided Amplification and CRISPR-Cas12a with Rotated Magnetic Extraction for Salmonella Detection

Background: Foodborne pathogenic bacteria threaten worldwide public health, and simple bacterial detection methods are in urgent need. Here, we established a lab-on-a-tube biosensor for simple, rapid, sensitive, and specific detection of foodborne bacteria. Methods: A rotatable Halbach cylinder magn...

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Autores principales: Wu, Shangyi, Yuan, Jing, Xu, Ai, Wang, Lei, Li, Yanbin, Lin, Jianhan, Yue, Xiqing, Xi, Xinge
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10144542/
https://www.ncbi.nlm.nih.gov/pubmed/37421063
http://dx.doi.org/10.3390/mi14040830
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author Wu, Shangyi
Yuan, Jing
Xu, Ai
Wang, Lei
Li, Yanbin
Lin, Jianhan
Yue, Xiqing
Xi, Xinge
author_facet Wu, Shangyi
Yuan, Jing
Xu, Ai
Wang, Lei
Li, Yanbin
Lin, Jianhan
Yue, Xiqing
Xi, Xinge
author_sort Wu, Shangyi
collection PubMed
description Background: Foodborne pathogenic bacteria threaten worldwide public health, and simple bacterial detection methods are in urgent need. Here, we established a lab-on-a-tube biosensor for simple, rapid, sensitive, and specific detection of foodborne bacteria. Methods: A rotatable Halbach cylinder magnet and an iron wire netting with magnetic silica beads (MSBs) were used for simple and effective extraction and purification of DNA from the target bacteria, and recombinase-aided amplification (RAA) was combined with clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins12a(CRISPR-Cas12a) to amplify DNA and generate fluorescent signal. First, 15 mL of the bacterial sample was centrifuged, and the bacterial pellet was lysed by protease to release target DNA. Then, DNA-MSB complexes were formed as the tube was intermittently rotated and distributed uniformly onto the iron wire netting inside the Halbach cylinder magnet. Finally, the purified DNA was amplified using RAA and quantitatively detected by the CRISPR-Cas12a assay. Results: This biosensor could quantitatively detect Salmonella in spiked milk samples in 75 min, with a lower detection limit of 6 CFU/mL. The fluorescent signal of 10(2) CFU/mL Salmonella Typhimurium was over 2000 RFU, while 10(4) CFU/mL Listeria monocytogenes, Bacillus cereus, and E. coli O157:H7 were selected as non-target bacteria and had signals less than 500 RFU (same as the negative control). Conclusions: This lab-on-a-tube biosensor integrates cell lysis, DNA extraction, and RAA amplification in one 15 mL tube to simplify the operation and avoid contamination, making it suitable for low-concentration Salmonella detection.
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spelling pubmed-101445422023-04-29 A Lab-on-a-Tube Biosensor Combining Recombinase-Aided Amplification and CRISPR-Cas12a with Rotated Magnetic Extraction for Salmonella Detection Wu, Shangyi Yuan, Jing Xu, Ai Wang, Lei Li, Yanbin Lin, Jianhan Yue, Xiqing Xi, Xinge Micromachines (Basel) Article Background: Foodborne pathogenic bacteria threaten worldwide public health, and simple bacterial detection methods are in urgent need. Here, we established a lab-on-a-tube biosensor for simple, rapid, sensitive, and specific detection of foodborne bacteria. Methods: A rotatable Halbach cylinder magnet and an iron wire netting with magnetic silica beads (MSBs) were used for simple and effective extraction and purification of DNA from the target bacteria, and recombinase-aided amplification (RAA) was combined with clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins12a(CRISPR-Cas12a) to amplify DNA and generate fluorescent signal. First, 15 mL of the bacterial sample was centrifuged, and the bacterial pellet was lysed by protease to release target DNA. Then, DNA-MSB complexes were formed as the tube was intermittently rotated and distributed uniformly onto the iron wire netting inside the Halbach cylinder magnet. Finally, the purified DNA was amplified using RAA and quantitatively detected by the CRISPR-Cas12a assay. Results: This biosensor could quantitatively detect Salmonella in spiked milk samples in 75 min, with a lower detection limit of 6 CFU/mL. The fluorescent signal of 10(2) CFU/mL Salmonella Typhimurium was over 2000 RFU, while 10(4) CFU/mL Listeria monocytogenes, Bacillus cereus, and E. coli O157:H7 were selected as non-target bacteria and had signals less than 500 RFU (same as the negative control). Conclusions: This lab-on-a-tube biosensor integrates cell lysis, DNA extraction, and RAA amplification in one 15 mL tube to simplify the operation and avoid contamination, making it suitable for low-concentration Salmonella detection. MDPI 2023-04-09 /pmc/articles/PMC10144542/ /pubmed/37421063 http://dx.doi.org/10.3390/mi14040830 Text en © 2023 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
Wu, Shangyi
Yuan, Jing
Xu, Ai
Wang, Lei
Li, Yanbin
Lin, Jianhan
Yue, Xiqing
Xi, Xinge
A Lab-on-a-Tube Biosensor Combining Recombinase-Aided Amplification and CRISPR-Cas12a with Rotated Magnetic Extraction for Salmonella Detection
title A Lab-on-a-Tube Biosensor Combining Recombinase-Aided Amplification and CRISPR-Cas12a with Rotated Magnetic Extraction for Salmonella Detection
title_full A Lab-on-a-Tube Biosensor Combining Recombinase-Aided Amplification and CRISPR-Cas12a with Rotated Magnetic Extraction for Salmonella Detection
title_fullStr A Lab-on-a-Tube Biosensor Combining Recombinase-Aided Amplification and CRISPR-Cas12a with Rotated Magnetic Extraction for Salmonella Detection
title_full_unstemmed A Lab-on-a-Tube Biosensor Combining Recombinase-Aided Amplification and CRISPR-Cas12a with Rotated Magnetic Extraction for Salmonella Detection
title_short A Lab-on-a-Tube Biosensor Combining Recombinase-Aided Amplification and CRISPR-Cas12a with Rotated Magnetic Extraction for Salmonella Detection
title_sort lab-on-a-tube biosensor combining recombinase-aided amplification and crispr-cas12a with rotated magnetic extraction for salmonella detection
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10144542/
https://www.ncbi.nlm.nih.gov/pubmed/37421063
http://dx.doi.org/10.3390/mi14040830
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