An Inexpensive CRISPR-Based Point-of-Care Test for the Identification of Meat Species and Meat Products
Abstract
:1. Introduction
2. Materials and Methods
2.1. Sample Preparation
2.2. PCR and LAMP Primers Design
2.3. Species-Specific CRISPR RNA (crRNAs) Design
2.4. Assessment of crRNAs and LAMP Primers for Intra-Specific Conservation
2.5. In Vitro RNA Transcription Using T7 RNA Polymerase
2.6. LAMP-CRISPR/Cas12a Fluorescence Assay
2.7. LAMP-CRISPR/Cas12a Colorimetric Assay
2.8. Sensitivity and Specificity of the LAMP-CRISPR/Cas12a Colorimetric Assay
2.9. LAMP-CRISPR/Cas12a with Lateral Flow Dipstick (LFD) Assay
2.10. On-Site Application of the LAMP-CRISPR/Cas12a Assay
3. Results
3.1. CRISPR/Cas12a crRNA Activity
3.2. Sensitivity Evaluation of the LAMP-CRISPR/Cas12a Fluorescence Assay
3.3. Establishment and Specificity Assessment of the LAMP-CRISPR/Cas12a Colorimetric Meat Detection Assay
3.4. Colorimetric and Lateral Flow Detection in Mixed-Meat Products
3.5. On-Site Application of the CRISPR/Cas12a Colorimetric Assay
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
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Tao, D.; Xiao, X.; Lan, X.; Xu, B.; Wang, Y.; Khazalwa, E.M.; Pan, W.; Ruan, J.; Jiang, Y.; Liu, X.; et al. An Inexpensive CRISPR-Based Point-of-Care Test for the Identification of Meat Species and Meat Products. Genes 2022, 13, 912. https://doi.org/10.3390/genes13050912
Tao D, Xiao X, Lan X, Xu B, Wang Y, Khazalwa EM, Pan W, Ruan J, Jiang Y, Liu X, et al. An Inexpensive CRISPR-Based Point-of-Care Test for the Identification of Meat Species and Meat Products. Genes. 2022; 13(5):912. https://doi.org/10.3390/genes13050912
Chicago/Turabian StyleTao, Dagang, Xiao Xiao, Xiaochen Lan, Bingrong Xu, Yuan Wang, Emmanuel Mulaya Khazalwa, Wenya Pan, Jinxue Ruan, Yu Jiang, Xiangdong Liu, and et al. 2022. "An Inexpensive CRISPR-Based Point-of-Care Test for the Identification of Meat Species and Meat Products" Genes 13, no. 5: 912. https://doi.org/10.3390/genes13050912