Rapid Visual Detection of African Swine Fever Virus with a CRISPR/Cas12a Lateral Flow Strip Based on Structural Protein Gene D117L
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Reagents and Viruses
2.2. Cloning of LbCas12a Gene and Protein Expression and Purification
2.3. Detection of Endonuclease Activity of the Purified LbCas12a Protein
2.4. Design of ASFV Structural Protein p17 Gene (D117L) crRNA and Its Mediated CRISPR/Cas12a Reaction
2.5. Recombinant Polymerase Amplification (RPA)
2.6. Establishment of RPA-CRISPR/Cas12a-Lateral Flow Strip (LFS) Detection Method
2.7. Sensitivity and Specificity of D117L-RPA-Cas12a-LFS Detection Method
2.8. Clinical Sample Processing and Nucleic Acid Extraction
3. Results
3.1. CRISPR/Cas12a Reaction Mediated by ASFV Structural Protein Gene D117L crRNA1 and Its Optimization
3.2. The Detection Sensitivity of the CRISPR/Cas12a Reaction
3.3. Establishment of CRISPR/Cas12a Mediated Lateral Flow Strip (LFS) Method, Its Sensitivity, and Its Specificity
3.4. Detection of Clinical Samples by RPA-CRISPR/Cas12a-LFS
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
References
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Sample Types | Sample Numbers | Results (Positive/Negative) | |
---|---|---|---|
RPA-Cas12a-LFS | qPCR | ||
Heart | 2 | 1/1 | 1/1 |
Liver | 4 | 2/2 | 2/2 |
Spleen | 4 | 2/2 | 2/2 |
Lung | 6 | 2/4 | 2/4 |
Kidney | 4 | 2/2 | 2/2 |
Oral swab | 2 | 0/2 | 0/2 |
Blood | 26 | 9/17 | 9/17 |
Serum | 20 | 3/17 | 3/17 |
Total | 68 | 21/47 | 21/47 |
Positive rates | 44.7% | 44.7% |
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Zhang, D.; Jiang, S.; Xia, N.; Zhang, Y.; Zhang, J.; Liu, A.; Zhang, C.; Chen, N.; Meurens, F.; Zheng, W.; et al. Rapid Visual Detection of African Swine Fever Virus with a CRISPR/Cas12a Lateral Flow Strip Based on Structural Protein Gene D117L. Animals 2023, 13, 3712. https://doi.org/10.3390/ani13233712
Zhang D, Jiang S, Xia N, Zhang Y, Zhang J, Liu A, Zhang C, Chen N, Meurens F, Zheng W, et al. Rapid Visual Detection of African Swine Fever Virus with a CRISPR/Cas12a Lateral Flow Strip Based on Structural Protein Gene D117L. Animals. 2023; 13(23):3712. https://doi.org/10.3390/ani13233712
Chicago/Turabian StyleZhang, Desheng, Sen Jiang, Nengwen Xia, Youwen Zhang, Jiajia Zhang, Anjing Liu, Chenyang Zhang, Nanhua Chen, Francois Meurens, Wanglong Zheng, and et al. 2023. "Rapid Visual Detection of African Swine Fever Virus with a CRISPR/Cas12a Lateral Flow Strip Based on Structural Protein Gene D117L" Animals 13, no. 23: 3712. https://doi.org/10.3390/ani13233712