Aptamer-CRISPR/Cas12a-Based Lateral Flow Technique for Visualized Rapid Detection of Endogenous Damage Factor Neu5Gc in Red Meat
Abstract
1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Aptamer Truncation Optimization
2.3. Aptamer Optimization Selection and crRNA Design
2.4. Construction of Aptamer-Cas12a Test Strip System and Validation of Design Principles
2.5. Aptamer-Cas12a Test Strip System Optimization
2.6. Specificity Determination of Neu5Gc Lateral Flow Detection Test Strips Based on Aptamer-Cas12a
2.7. Aptamer-Cas12a Test Strip Sensitivity Assay
2.8. Aptamer-Cas12a-Based Neu5Gc Lateral Flow Test Strip Spiking and Actual Sample Detection
3. Results
3.1. Analysis of Truncation Optimization of Nucleic Acid Aptamers
3.2. Proof of Principle Validation of Neu5Gc Lateral Flow Detection Test Strips Based on Aptamer-Cas12a
3.3. Optimization Results of Aptamer-Cas12a Test Strip Probe Concentration and Aptamer Content
3.4. Aptamer-Cas12a Test Strip Reaction Time and Temperature Optimization Results
3.5. Based on the Specific Determination Results of the Lateral Flow Test Strip for Neu5Gc Using the Aptamer-Cas12a System
3.6. Aptamer-Cas12a Test Strip Sensitivity Assay Results
3.7. Results of Neu5Gc Lateral Flow Test Strip Spiking Assay Based on Aptamer-Cas12a
3.8. Actual Sample Detection of Neu5Gc Lateral Flow Test Strips Based on Aptamer-Cas12a
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Sequence Name | Sequences (5’–3’) | Sequence Lengths |
---|---|---|
N2A9.81 | CATCTGCAGTGTGGCACCATGGGCATTGGGGGGCGAGGGTGGTTGGAGCGTTTGTGTGCGACGTGCTGAGCGTGAATTCGC | 81 bp |
N2A9.78 | CATCTGCAGTGTGGCACCATGGGCATTGGGGGGCGAGGGTGGTTGGAGCGTTTGTGTGCGACGTGCTGAGCGTGAATTCGC | 78 bp |
N2A9.70 | CATCTGCAGTGTGGCACCATGGGCATTGGGGGGCGAGGGTGGTTGGAGCGTTTGTGTGCGACGTGCTGAGCGTGAATTCGC | 70 bp |
N2A9.61 | CATCTGCAGTGTGGCACCATGGGCATTGGGGGGCGAGGGTGGTTGGAGCGTTTGTGTGCGACGTGCTGAGCGTGAATTCGC | 61 bp |
N2A9.55 | CATCTGCAGTGTGGCACCATGGGCATTGGGGGGCGAGGGTGGTTGGAGCGTTTGTGTGCGACGTGCTGAGCGTGAATTCGC | 55 bp |
Remark | Underscores are truncated removal sequences |
Reaction | System Name | Volumetric (µL) |
---|---|---|
10 min at room temperature | Liquid to be examined | 5(0) |
Aptamer100 nM | 5 | |
CRISPR/Cas12a reaction 37 °C for 15 min | Buffer | 5 |
LbCas12a, 1 µM | 5 | |
crRNA, 1 µM | 5 | |
Dual-labeled probes, 5 µM | 5 | |
DEPC water | 20(25) | |
Total | 50 |
Detection Method | Detection Time | Detection Temperature | Minimum Detection Limit | Required Instrument |
---|---|---|---|---|
HPLC [41] | 10 h | 18–25 °C | 0.02 ng/μL | High Performance Liquid Chromatography |
MS [42] | 12 h | 18–25 °C | 0.02pg/μL | Mass spectrometer |
Aptamer-ELISA [22] | 17 h | 37 °C | 0.71 ng/mL | microplate reader |
Aptamer-Cas12a test strip | 25 min | 37 °C | 10 ng/mL | Metal bath or Warm water bath |
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Guo, Y.; Ren, H.; Wang, H.; Duan, X.; Qi, S.; Yang, X.; Shangguan, C.; Li, H.; Li, Y.; Hu, P.; et al. Aptamer-CRISPR/Cas12a-Based Lateral Flow Technique for Visualized Rapid Detection of Endogenous Damage Factor Neu5Gc in Red Meat. Foods 2025, 14, 2879. https://doi.org/10.3390/foods14162879
Guo Y, Ren H, Wang H, Duan X, Qi S, Yang X, Shangguan C, Li H, Li Y, Hu P, et al. Aptamer-CRISPR/Cas12a-Based Lateral Flow Technique for Visualized Rapid Detection of Endogenous Damage Factor Neu5Gc in Red Meat. Foods. 2025; 14(16):2879. https://doi.org/10.3390/foods14162879
Chicago/Turabian StyleGuo, Yuxi, Honglin Ren, Han Wang, Xuepeng Duan, Shuaihao Qi, Xi Yang, Chunyi Shangguan, Haosong Li, Yansong Li, Pan Hu, and et al. 2025. "Aptamer-CRISPR/Cas12a-Based Lateral Flow Technique for Visualized Rapid Detection of Endogenous Damage Factor Neu5Gc in Red Meat" Foods 14, no. 16: 2879. https://doi.org/10.3390/foods14162879
APA StyleGuo, Y., Ren, H., Wang, H., Duan, X., Qi, S., Yang, X., Shangguan, C., Li, H., Li, Y., Hu, P., Lu, Q., & Lu, S. (2025). Aptamer-CRISPR/Cas12a-Based Lateral Flow Technique for Visualized Rapid Detection of Endogenous Damage Factor Neu5Gc in Red Meat. Foods, 14(16), 2879. https://doi.org/10.3390/foods14162879