A Lab-on-a-Tube Biosensor Combining Recombinase-Aided Amplification and CRISPR-Cas12a with Rotated Magnetic Extraction for <i>Salmonella</i> 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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| Format: | Article |
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MDPI AG
2023-04-01
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| Series: | Micromachines |
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| Online Access: | https://www.mdpi.com/2072-666X/14/4/830 |
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| author | Shangyi Wu Jing Yuan Ai Xu Lei Wang Yanbin Li Jianhan Lin Xiqing Yue Xinge Xi |
| author_facet | Shangyi Wu Jing Yuan Ai Xu Lei Wang Yanbin Li Jianhan Lin Xiqing Yue Xinge Xi |
| author_sort | Shangyi Wu |
| collection | DOAJ |
| 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 <i>Salmonella</i> in spiked milk samples in 75 min, with a lower detection limit of 6 CFU/mL. The fluorescent signal of 10<sup>2</sup> CFU/mL <i>Salmonella</i> Typhimurium was over 2000 RFU, while 10<sup>4</sup> CFU/mL <i>Listeria</i> monocytogenes, <i>Bacillus</i> cereus, and <i>E. coli</i> 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 <i>Salmonella</i> detection. |
| first_indexed | 2024-03-11T04:43:28Z |
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| institution | Directory Open Access Journal |
| issn | 2072-666X |
| language | English |
| last_indexed | 2024-03-11T04:43:28Z |
| publishDate | 2023-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Micromachines |
| spelling | doaj.art-513031e718de459e9707f38bf8bfc1a62023-11-17T20:29:59ZengMDPI AGMicromachines2072-666X2023-04-0114483010.3390/mi14040830A Lab-on-a-Tube Biosensor Combining Recombinase-Aided Amplification and CRISPR-Cas12a with Rotated Magnetic Extraction for <i>Salmonella</i> DetectionShangyi Wu0Jing Yuan1Ai Xu2Lei Wang3Yanbin Li4Jianhan Lin5Xiqing Yue6Xinge Xi7College of Food Science, Shenyang Agricultural University, Shenyang 110866, ChinaKey Laboratory of Agricultural Information Acquisition Technology, Ministry of Agriculture and Rural Affairs, China Agricultural University, Beijing 100083, ChinaKey Laboratory of Agricultural Information Acquisition Technology, Ministry of Agriculture and Rural Affairs, China Agricultural University, Beijing 100083, ChinaKey Laboratory of Agricultural Information Acquisition Technology, Ministry of Agriculture and Rural Affairs, China Agricultural University, Beijing 100083, ChinaDepartment of Biological and Agricultural Engineering, University of Arkansas, Fayetteville, AR 72701, USAKey Laboratory of Agricultural Information Acquisition Technology, Ministry of Agriculture and Rural Affairs, China Agricultural University, Beijing 100083, ChinaCollege of Food Science, Shenyang Agricultural University, Shenyang 110866, ChinaKey Laboratory of Agricultural Information Acquisition Technology, Ministry of Agriculture and Rural Affairs, China Agricultural University, Beijing 100083, ChinaBackground: 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 <i>Salmonella</i> in spiked milk samples in 75 min, with a lower detection limit of 6 CFU/mL. The fluorescent signal of 10<sup>2</sup> CFU/mL <i>Salmonella</i> Typhimurium was over 2000 RFU, while 10<sup>4</sup> CFU/mL <i>Listeria</i> monocytogenes, <i>Bacillus</i> cereus, and <i>E. coli</i> 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 <i>Salmonella</i> detection.https://www.mdpi.com/2072-666X/14/4/830lab-on-a-tube biosensorlarge-volume sampleRAA/ CRISPR-Cas12arotated magnetic extraction<i>Salmonella</i> detection |
| spellingShingle | Shangyi Wu Jing Yuan Ai Xu Lei Wang Yanbin Li Jianhan Lin Xiqing Yue Xinge Xi A Lab-on-a-Tube Biosensor Combining Recombinase-Aided Amplification and CRISPR-Cas12a with Rotated Magnetic Extraction for <i>Salmonella</i> Detection Micromachines lab-on-a-tube biosensor large-volume sample RAA/ CRISPR-Cas12a rotated magnetic extraction <i>Salmonella</i> detection |
| title | A Lab-on-a-Tube Biosensor Combining Recombinase-Aided Amplification and CRISPR-Cas12a with Rotated Magnetic Extraction for <i>Salmonella</i> Detection |
| title_full | A Lab-on-a-Tube Biosensor Combining Recombinase-Aided Amplification and CRISPR-Cas12a with Rotated Magnetic Extraction for <i>Salmonella</i> Detection |
| title_fullStr | A Lab-on-a-Tube Biosensor Combining Recombinase-Aided Amplification and CRISPR-Cas12a with Rotated Magnetic Extraction for <i>Salmonella</i> Detection |
| title_full_unstemmed | A Lab-on-a-Tube Biosensor Combining Recombinase-Aided Amplification and CRISPR-Cas12a with Rotated Magnetic Extraction for <i>Salmonella</i> Detection |
| title_short | A Lab-on-a-Tube Biosensor Combining Recombinase-Aided Amplification and CRISPR-Cas12a with Rotated Magnetic Extraction for <i>Salmonella</i> Detection |
| title_sort | lab on a tube biosensor combining recombinase aided amplification and crispr cas12a with rotated magnetic extraction for i salmonella i detection |
| topic | lab-on-a-tube biosensor large-volume sample RAA/ CRISPR-Cas12a rotated magnetic extraction <i>Salmonella</i> detection |
| url | https://www.mdpi.com/2072-666X/14/4/830 |
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