CRISPR-Cas12a/Aurora Deoxyribozyme Cascade: A Label-Free Ultrasensitive Platform for Rapid <i>Salmonella</i> Detection
The rapid and ultrasensitive detection of <i>Salmonella</i> holds strategic significance for food safety surveillance and public health protection systems. This study innovatively developed a label-free biosensing platform based on the synergistic integration of Clustered Regularly Inter...
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2025-05-01
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| Series: | Foods |
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| author | Cong Shi Huimin Tan Zhou Yu Weilin Li Yan Man Qinghai Zhang |
| author_facet | Cong Shi Huimin Tan Zhou Yu Weilin Li Yan Man Qinghai Zhang |
| author_sort | Cong Shi |
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| description | The rapid and ultrasensitive detection of <i>Salmonella</i> holds strategic significance for food safety surveillance and public health protection systems. This study innovatively developed a label-free biosensing platform based on the synergistic integration of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas12a and the fluorescent deoxyribozyme Aurora for the efficient detection of foodborne Salmonella. The detection mechanism operates through a molecular cascade reaction: target-activated Cas12a protein specifically degrades Aurora deoxyribozyme via its trans-cleavage activity, thereby abolishing the enzyme’s catalytic capability to convert 4-methylumbelliferyl phosphate (4-MUP) into the highly fluorescent product 4-methylumbelliferone (4-MU). This cascade ultimately enables quantitative target analysis through fluorescence signal attenuation. Following systematic optimization of critical reaction parameters, the biosensing system demonstrated exceptional analytical performance: a detection limit of 1.29 CFU/mL with excellent linearity (R<sup>2</sup> = 0.992) spanning six orders of magnitude (1.65 × 10<sup>1</sup>–10<sup>6</sup> CFU/mL), along with high specificity against multiple interfering bacterial strains. Spike-and-recovery tests in complex food matrices (milk, chicken, and lettuce) yielded recoveries of 90.91–99.40% (RSD = 3.55–4.72%), confirming robust practical applicability. Notably, the platform design allows flexible detection of other pathogens through simple replacement of CRISPR guide sequences. |
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| publishDate | 2025-05-01 |
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| spelling | doaj-art-9d067fc99d174b82b6cebb83b413cbad2025-08-20T02:33:11ZengMDPI AGFoods2304-81582025-05-011411189210.3390/foods14111892CRISPR-Cas12a/Aurora Deoxyribozyme Cascade: A Label-Free Ultrasensitive Platform for Rapid <i>Salmonella</i> DetectionCong Shi0Huimin Tan1Zhou Yu2Weilin Li3Yan Man4Qinghai Zhang5Key Laboratory of Environmental Pollution Monitoring and Disease Control, Ministry of Education, School of Public Health, Guizhou Medical University, No. 6 Ankang Road, Guian New Area, Guiyang 561113, ChinaKey Laboratory of Environmental Pollution Monitoring and Disease Control, Ministry of Education, School of Public Health, Guizhou Medical University, No. 6 Ankang Road, Guian New Area, Guiyang 561113, ChinaKey Laboratory of Environmental Pollution Monitoring and Disease Control, Ministry of Education, School of Public Health, Guizhou Medical University, No. 6 Ankang Road, Guian New Area, Guiyang 561113, ChinaKey Laboratory of Environmental Pollution Monitoring and Disease Control, Ministry of Education, School of Public Health, Guizhou Medical University, No. 6 Ankang Road, Guian New Area, Guiyang 561113, ChinaKey Laboratory of Environmental Pollution Monitoring and Disease Control, Ministry of Education, School of Public Health, Guizhou Medical University, No. 6 Ankang Road, Guian New Area, Guiyang 561113, ChinaKey Laboratory of Environmental Pollution Monitoring and Disease Control, Ministry of Education, School of Public Health, Guizhou Medical University, No. 6 Ankang Road, Guian New Area, Guiyang 561113, ChinaThe rapid and ultrasensitive detection of <i>Salmonella</i> holds strategic significance for food safety surveillance and public health protection systems. This study innovatively developed a label-free biosensing platform based on the synergistic integration of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas12a and the fluorescent deoxyribozyme Aurora for the efficient detection of foodborne Salmonella. The detection mechanism operates through a molecular cascade reaction: target-activated Cas12a protein specifically degrades Aurora deoxyribozyme via its trans-cleavage activity, thereby abolishing the enzyme’s catalytic capability to convert 4-methylumbelliferyl phosphate (4-MUP) into the highly fluorescent product 4-methylumbelliferone (4-MU). This cascade ultimately enables quantitative target analysis through fluorescence signal attenuation. Following systematic optimization of critical reaction parameters, the biosensing system demonstrated exceptional analytical performance: a detection limit of 1.29 CFU/mL with excellent linearity (R<sup>2</sup> = 0.992) spanning six orders of magnitude (1.65 × 10<sup>1</sup>–10<sup>6</sup> CFU/mL), along with high specificity against multiple interfering bacterial strains. Spike-and-recovery tests in complex food matrices (milk, chicken, and lettuce) yielded recoveries of 90.91–99.40% (RSD = 3.55–4.72%), confirming robust practical applicability. Notably, the platform design allows flexible detection of other pathogens through simple replacement of CRISPR guide sequences.https://www.mdpi.com/2304-8158/14/11/1892nucleic acid detectionfluorescence sensingfood safetyDNAzymepathogen identification |
| spellingShingle | Cong Shi Huimin Tan Zhou Yu Weilin Li Yan Man Qinghai Zhang CRISPR-Cas12a/Aurora Deoxyribozyme Cascade: A Label-Free Ultrasensitive Platform for Rapid <i>Salmonella</i> Detection Foods nucleic acid detection fluorescence sensing food safety DNAzyme pathogen identification |
| title | CRISPR-Cas12a/Aurora Deoxyribozyme Cascade: A Label-Free Ultrasensitive Platform for Rapid <i>Salmonella</i> Detection |
| title_full | CRISPR-Cas12a/Aurora Deoxyribozyme Cascade: A Label-Free Ultrasensitive Platform for Rapid <i>Salmonella</i> Detection |
| title_fullStr | CRISPR-Cas12a/Aurora Deoxyribozyme Cascade: A Label-Free Ultrasensitive Platform for Rapid <i>Salmonella</i> Detection |
| title_full_unstemmed | CRISPR-Cas12a/Aurora Deoxyribozyme Cascade: A Label-Free Ultrasensitive Platform for Rapid <i>Salmonella</i> Detection |
| title_short | CRISPR-Cas12a/Aurora Deoxyribozyme Cascade: A Label-Free Ultrasensitive Platform for Rapid <i>Salmonella</i> Detection |
| title_sort | crispr cas12a aurora deoxyribozyme cascade a label free ultrasensitive platform for rapid i salmonella i detection |
| topic | nucleic acid detection fluorescence sensing food safety DNAzyme pathogen identification |
| url | https://www.mdpi.com/2304-8158/14/11/1892 |
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